Production of glcnac containing bioproducts in a cell

ABSTRACT

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a method for the production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end by a cell as well as the purification of the di- or oligosaccharide from the cultivation. Furthermore, the disclosure provides a cell metabolically engineered for production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/EP2021/072269, filed Aug. 10, 2021,designating the United States of America and published as InternationalPatent Publication WO 2022/034075 A1 on Feb. 17, 2022, which claims thebenefit under Article 8 of the Patent Cooperation Treaty to EuropeanPatent Application Serial No. 20190206.1, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190198.0, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190200.4, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190201.2, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190202.0, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190203.8, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190204.6, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190205.3, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190207.9, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 20190208.7, filed Aug. 10, 2020, EuropeanPatent Application Serial No. 21168997.1, filed Apr. 16, 2021, EuropeanPatent Application Serial No. 21186202.4, filed Jul. 16, 2021, andEuropean Patent Application Serial No. 21186203.2, filed Jul. 16, 2021.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. § 1.821(c) or (e), a Sequence Listing ASCII textfile entitled “4006-P17265US (026-PCT-US) US Sequence Listing_ST25.txt,”103,605 bytes in size, generated Jan. 17, 2023, has been submitted, thecontents of which are hereby incorporated by reference.

TECHNICAL FIELD

This disclosure is in the technical field of synthetic biology andmetabolic engineering. More particularly, the disclosure is in thetechnical field of cultivation or fermentation of metabolicallyengineered cells. The disclosure describes a method for the productionof a di- or oligosaccharide with an N-acetylglucosamine unit at thereducing end by a cell as well as the purification of the di- oroligosaccharide from the cultivation. Furthermore, the disclosureprovides a cell metabolically engineered for production of a di- oroligosaccharide with an N-acetylglucosamine unit at the reducing end.

BACKGROUND

Carbohydrates, often present as glyco-conjugated forms to proteins andlipids, are involved in many vital phenomena such as differentiation,development and biological recognition processes related to thedevelopment and progress of fertilization, embryogenesis, inflammation,metastasis and host pathogen adhesion. Carbohydrates can also be presentas unconjugated glycans in body fluids and human milk wherein they alsomodulate important developmental and immunological processes (Bode,Early Hum. Dev. 1-4 (2015); Reily et al., Nat. Rev. Nephrol. 15, 346-366(2019); Varki, Glycobiology 27, 3-49 (2017)).

The di-saccharide Galβ1,3GlcNAc, also known as lacto-N-biose, LNB,N-acetyllactosamine type 1 or LacNAc type 1, is composed of a galactosewhich is beta-1,3 linked to an N-acetylglucosamine (GlcNAc). GlcNAc ispresent at the reducing end of the disaccharide. LNB is a well-knownprecursor of several important blood group epitopes, such as Lewis A,Lewis B, or sialyl Lewis A. LacNAc type 1 containing glycans play alsoan important role in tumour metastasis and are, therefore, considered astumour markers (Fischöder et al., Molecules 22, 1320 (2017)). They arealso important constituents of mucin-type glycoproteins. The ubiquitousdi-saccharide Galβ1,4GlcNAc, also known as N-acetyllactosamine, LacNAcor LacNAc type 2, is composed of a galactose which is beta-1,4 linked toan N-acetylglucosamine (GlcNAc). Also here, GlcNAc is present at thereducing end of the disaccharide. LacNAc is often over-expressed on thesurface of cancer cells where it is bound by tumour secreted galectinscontributing to cancer-related processes such as metastasis, adhesion,tumour survival, and immune escape (Romano and Oscarson, Org. Biomol.Chem. 17, 2265-2278 (2019)). LacNAc is also part of Lewis X, Lewis Y andsialyl Lewis X epitopes. Dimeric and elongated LacNAc structures of bothtype 1 (Galβ1,3GlcNAc) and type 2 (Galβ1,4GlcNAc) as well aspoly-N-acetyllactosamines (poly-LacNAc) have been frequently associatedwith inflammation processes and cancer. The di-saccharides LNB andLacNAc as well as moieties thereof and Lewis type epitopes also occur inhuman milk, as part of the human milk oligosaccharide (HMO) composition(Prudden et al., PNAS USA 114, 6954-6959 (2017)). Some of theBifidobacterium species present in the colon are able to specificallyconsume HMOs like LNB or LNB containing saccharides. Certainlactobacilli such as L. casei have been shown to metabolize the LacNAcdi-saccharide present in human milk in the early stage of lactation(Bidart et al., Sci. Rep. 8, 7152 (2018)). Consequently, lactobacilliand bifidobacteria represent up to 90% of the total gut flora inbreastfed infants (Fischöder et al., Molecules 22, 1320 (2017)).

Due to the plethora of important processes these di- andoligosaccharides with a GlcNAc at their reducing end are involved in,there is a large pharmaceutical and nutraceutical interest in developingnovel N-acetyllactosamine-based (type 1 or type 2) therapeutic agents orbeneficial nutritional compounds. Considerable effort is put intodeveloping synthesis processes for glycans like the di-saccharides LNBand LacNAc or for glycans containing LNB or LAcNAc at their reducingend.

Chemical synthesis methods are laborious and time-consuming and becauseof the large number of steps involved they are difficult to scale-up.Biocatalytic approaches offer many advantages and thus, an extensivenumber of publications related to enzymatic production of LNB, LacNAcand variables thereof are available. Bayón et al. (RSC Advances 3(30)(2013)) reported the usage of a purified β-Gal-3 galactosidase fromBacillus circulans to produce LNB from biomass with supplementedp-NP-Gal as donor and supplemented GlcNAc as acceptor. Patentapplication JP2017195793A describes another galactosidase, recombinantlyproduced by and purified from a Bacillus species, to be used in the invitro synthesis of galacto-oligosaccharides like LNB via hydrolysis.Other papers make use of LNB phosphorylase from Bifidobacteria toproduce LNB in enzymatic reaction mixtures starting from sucrose andsupplemented GlcNAc, additionally supplemented with sucrosephosphorylase, UDP-glucose-hexose-1-phosphate uridylyltransferase, andUDP-glucose 4-epimerase (Nishimoto and Kitaoka, Biosci. Biotechnol.Biochem. 71, 2101-2104 (2007); Nishimoto, Biosci. Biotechnol. Biochem.84, 17-24 (2020); US2010120096A; JP4264742 B2; JP2005341883 A2). Also,the use of in vitro reaction mixtures containing galactose, GlcNAc,galactokinase together with LNB phosphorylase has been presented(CN110527704 A). Another mode of action frequently reported makes use ofbacterial coupling to perform the catalytic conversions. In suchexamples, several recombinant bacterial strains expressing enzymesinvolved in the catalytic pathway to the saccharides of disclosure, weregrown and lysed to obtain the enzymes necessary in the catabolicreaction mixtures. In patent application JP2013201913, it describes thecoupled use of a Bifidobacterium breve MCC1320 or Bifidobacteriuminfantis strain together with a B. longum strain to express a LNBphosphorylase and a sucrose phosphorylase, respectively, in a mixturecontaining supplemented sucrose, GlcNAc, phosphate, UDP-glucose and/orUDP-galactose to make LNB. Endo and co-workers even reported the coupleduse of three bacterial strains, i.e., two recombinant E. coli strainsfor over-expression of galT, galK, galU, ppa and lgtB from Neisseriagonorrhoeae together with a Corynebacterium ammoniagenes strain for UTPproduction, to make LacNAc in a reaction supplemented with galactose andGlcNAc (Endo et al., Carb. Res. 316(1-4), 179-183 (1999)). The samegroup described a comparable coupling system whereby one of the E. colistrains expressed a beta1,4-galT from H. pylori instead of the NgLgtB totake part in the enzymatic conversions, supplemented with additionalGlcNAc, to produce LacNAc (Endo et al., Glycobiology 10(8), 809-813(2000)). The NmLgtB enzyme from N. meningitidis (Wakarchuk et al.,Protein Engineering, Design and Selection 11(4), 295-302 (1998)) hasalso been frequently cloned as part of a fusion protein together withgalE from E. coli or from Streptococcus thermophilus for the synthesisof LacNAc-based oligosaccharides in reactions with additional GlcNAc(Blixt et al., J. Org. Chem. 66(7), 2442-2448 (2001); Ruffing et al.,Metab. Eng. 8(5), 465-473 (2006); Mao et al., Biotechnol. Prog. 22(2),369-374 (2006)). Bacterial coupling has also been described in asynthesis method for the Lewis X epitope, also known as 3′-fucosylatedLacNAc (Koizumi et al., J. Ind. Microbiol. Biotech. 25, 213-217 (2000)).

Above methods often suffer from the relatively poor availability and/orstability of glycosyltransferases and/or glycosyl hydrolases, the demandfor optimal stoichiometric balancing, the need to regenerate in situnucleotide-sugars, the addition of multiple reaction compounds likee.g., the acceptors comprising GlcNAc or oligosaccharides containingGlcNAc at their reducing end, and the need to grow different productionorganisms that each separately produce one or more enzymes or a fusionenzyme necessary in the catalytic conversions. The most importantobstacle of this list is the cellular synthesis of the principalacceptor comprising GlcNAc or oligosaccharides containing GlcNAc attheir reducing end. In the examples referred to, the GlcNAcmonosaccharide used for synthesis of LNB, LacNAc or oligosaccharideshaving a GlcNAc at their reducing end is being externally supplementedto the reactions or cells involved.

Bettler and co-workers described the production of the hexasaccharideβGal(1,4)[βGlcNAc(1,4)]₄GlcNAc that was built with a single cell withoutsupplementation of GlcNAc (Bettler et al., Glycoconj. J. 16, 205-212(1999)). This hexasaccharide had a GlcNAc unit at its reducing end and aterminal LacNAc moiety at its non-reducing end. For this, the NodCenzyme (a β1,4-GlcNAc-oligosaccharide synthase) from the bacterialAzorhizobium species was co-expressed with the LgtB enzyme (aβ(1,4)-galactosyltransferase) from N. meningitidis in a recombinant E.coli cell. However, in this example the chitin structure(GlcNAc-GlcNAc)_(n) present in this hexasaccharide and having a GlcNAcat its reducing end was produced by ligation of UDP-GlcNAc moieties.Bettler and co-workers also described the production of the xenoantigenGalα1,3Galβ1,4[βGlcNAc(1,4)]₄GlcNAc containing a GlcNAc at its reducingend in a similar system with a recombinant E. coli cell co-expressingNodC, LgtB and the bovine a1,3-galactosyltransferase GstA (Bettler etal., Biochem. Biophys. Res. Commun. 302(3), 620-624 (2003)). Again, thechitin structure and the LacNAc moiety present in the heptasaccharideare being built from UDP-GlcNAc moieties instead of using non-activatedGlcNAc. Deng and co-workers reported the successful production of GlcNAcwith a recombinant E. coli cell via fermentation (Deng et al., Metab.Eng. 7, 201-214 (2005); EP1576106. In the microbial system Deng andco-workers developed, GlcNAc is being produced extracellularly of an E.coli cell, more specifically in the periplasm of E. coli, viade-phosphorylation of GlcNAc-6-phosphate during export of the latterone. As such, the GlcNAc moiety obtained is not available any longer forintracellular conversion like further glycosylation which is needed tocreate the saccharides of current disclosure. Also, the processdescribed by Deng and co-workers requires a two-phase fed batch systemthat needs precise control to minimize inhibitory effects ofphosphorylate amino sugars onto the production host, which makes theprocess not of commercial interest to produce high titers of(extracellular) GlcNAc.

This disclosure overcomes the above-described problems as it provides amethod and a cell to produce the desired products in a relatively easyand if needed, continuous process.

BRIEF SUMMARY

Surprisingly, it has now been found that it is possible to produceGlcNAc by a single cell and to further glycosylate this GlcNAcmonosaccharide by the same cell to create a di- or oligosaccharidehaving a GlcNAc at its reducing end. The disclosure provides a methodfor the production of a di- or oligosaccharide with anN-acetylglucosamine unit at the reducing end by a cell. The methodcomprises the steps of providing a cell which is capable to synthesize anucleotide-sugar, to synthesize GlcNAc, and of glycosylating the GlcNAcmonosaccharide. The disclosure also relates to methods of producing adi- or oligosaccharide with a GlcNAc at the reducing end by cultivationthe cell under conditions permissive for producing the di- oroligosaccharide. Next, the disclosure also provides methods to separatethe di- or oligosaccharide from the cultivation. Furthermore, thedisclosure provides a cell metabolically engineered for production of adi- or oligosaccharide with an N-acetylglucosamine unit at the reducingend.

Definitions

The words used in this specification to describe the disclosure and itsvarious embodiments are to be understood not only in the sense of theircommonly defined meanings, but to include by special definition in thisspecification structure, material or acts beyond the scope of thecommonly defined meanings. Thus, if an element can be understood in thecontext of this specification as including more than one meaning, thenits use in a claim must be understood as being generic to all possiblemeanings supported by the specification and by the word itself.

The various embodiments and aspects of embodiments of the disclosuredescribed herein are to be understood not only in the order and contextspecifically described in this specification, but to include any orderand any combination thereof. Whenever the context requires, all wordsused in the singular number shall be deemed to include the plural andvice versa. Unless defined otherwise, all technical and scientific termsused herein generally have the same meaning as commonly understood byone of ordinary skill in the art to which this disclosure belongs.Generally, the nomenclature used herein and the laboratory procedures incell culture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described herein are those well-known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Generally, purification steps are performedaccording to the manufacturer's specifications.

In the specification, there have been disclosed embodiments of thedisclosure, and although specific terms are employed, the terms are usedin a descriptive sense only and not for purposes of limitation, thescope of the disclosure being set forth in the following claims. It mustbe understood that the illustrated embodiments have been set forth onlyfor the purposes of example and that it should not be taken as limitingthe disclosure. It will be apparent to those skilled in the art thatalterations, other embodiments, improvements, details and uses can bemade consistent with the letter and spirit of the disclosure herein andwithin the scope of this disclosure, which is limited only by theclaims, construed in accordance with the patent law, including thedoctrine of equivalents. In the claims which follow, referencecharacters used to designate claim steps are provided for convenience ofdescription only, and are not intended to imply any particular order forperforming the steps.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. Throughout the disclosure, the verb “to comprise” maybe replaced by “to consist” or “to consist essentially of” and viceversa. In addition the verb “to consist” may be replaced by “to consistessentially of” meaning that a composition as defined herein maycomprise additional component(s) than the ones specifically identified,the additional component(s) not altering the unique characteristic ofthe disclosure. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere is one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one.”

Throughout the disclosure, unless explicitly stated otherwise, thearticles “a” and “an” are preferably replaced by “at least two,” morepreferably by “at least three,” even more preferably by “at least four,”even more preferably by “at least five,” even more preferably by “atleast six,” most preferably by “at least two.”

Throughout the disclosure, unless explicitly stated otherwise, thefeatures “synthesize,” “synthesized” and “synthesis” are interchangeablyused with the features “produce,” “produced” and “production,”respectively.

Each embodiment as identified herein may be combined together unlessotherwise indicated. All publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference. The full content of the priorityapplications, including EP20190198, EP20190200, and EP20190206 are alsoincorporated by reference to the same extent as if the priorityapplication was specifically and individually indicated to beincorporated by reference.

According to the disclosure, the term “polynucleotide(s)” generallyrefers to any polyribonucleotide or polydeoxyribonucleotide, which maybe unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide(s)”include, without limitation, single- and double-stranded DNA, DNA thatis a mixture of single- and double-stranded regions or single-, double-and triple-stranded regions, single- and double-stranded RNA, and RNAthat is mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded regions, or a mixture of single- anddouble-stranded regions. In addition, “polynucleotide” as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term “polynucleotide(s)” alsoincludes DNAs or RNAs as described above that contain one or moremodified bases. Thus, DNAs or RNAs with backbones modified for stabilityor for other reasons are “polynucleotide(s)” according to thedisclosure. Moreover, DNAs or RNAs comprising unusual bases, such asinosine, or modified bases, such as tritylated bases, are to beunderstood to be covered by the term “polynucleotides.” It will beappreciated that a great variety of modifications have been made to DNAand RNA that serve many useful purposes known to those of skill in theart. The term “polynucleotide(s)” as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including, for example, simple andcomplex cells. The term “polynucleotide(s)” also embraces shortpolynucleotides often referred to as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to the skilled person. The same typeof modification may be present in the same or varying degree at severalsites in a given polypeptide. Furthermore, a given polypeptide maycontain many types of modifications. Modifications can occur anywhere ina polypeptide, including the peptide backbone, the amino acidsidechains, and the amino or carboxyl termini. Modifications include,for example, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, transfer-RNA mediated addition of aminoacids to proteins, such as arginylation, and ubiquitination.Polypeptides may be branched or cyclic, with or without branching.Cyclic, branched and branched circular polypeptides may result frompost-translational natural processes and may be made by entirelysynthetic methods, as well.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the disclosure. The term also encompasses polynucleotidesthat include a single continuous region or discontinuous regionsencoding the polypeptide (for example, interrupted by integrated phageor an insertion sequence or editing) together with additional regionsthat also may contain coding and/or non-coding sequences.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated,” as the term is employedherein. Similarly, a “synthetic” sequence, as the term is used herein,means any sequence that has been generated synthetically and notdirectly isolated from a natural source. “Synthesized,” as the term isused herein, means any synthetically generated sequence and not directlyisolated from a natural source.

The terms “recombinant” or “transgenic” or “metabolically engineered” or“genetically modified,” as used herein with reference to a cell or hostcell, are used interchangeably and indicates that the bacterial cellreplicates a heterologous nucleic acid, or expresses a peptide orprotein encoded by a heterologous nucleic acid (i.e., a sequence“foreign to the cell” or a sequence “foreign to the location orenvironment in the cell”). Such cells are described to be transformedwith at least one heterologous or exogenous gene, or are described to betransformed by the introduction of at least one heterologous orexogenous gene. Metabolically engineered or recombinant or transgeniccells can contain genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells can also containgenes found in the native form of the cell wherein the genes aremodified and re-introduced into the cell by artificial means. The termsalso encompass cells that contain a nucleic acid endogenous to the cellthat has been modified or its expression or activity has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, replacement of a promoter;site-specific mutation; and related techniques. Accordingly, a“recombinant polypeptide” is one which has been produced by arecombinant cell. A “heterologous sequence” or a “heterologous nucleicacid,” as used herein, is one that originates from a source foreign tothe particular cell (e.g., from a different species), or, if from thesame source, is modified from its original form or place in the genome.Thus, a heterologous nucleic acid operably linked to a promoter is froma source different from that from which the promoter was derived, or, iffrom the same source, is modified from its original form or place in thegenome. The heterologous sequence may be stably introduced, e.g., bytransfection, transformation, conjugation or transduction, into thegenome of the host microorganism cell, wherein techniques may be appliedwhich will depend on the cell and the sequence that is to be introduced.Various techniques are known to a person skilled in the art and are,e.g., disclosed in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). The term “mutant” cell or microorganism as usedwithin the context of the disclosure refers to a cell or microorganismwhich is genetically modified.

The terms “cell genetically modified for the production of a di- oroligosaccharide having an N-acetylglucosamine (GlcNAc) unit at thereducing end” within the context of the disclosure refers to a cell of amicroorganism which is genetically modified in the expression oractivity of one or more enzyme(s) selected from the group comprising:glucosamine 6-phosphate N-acetyltransferase, phosphatase,glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.

The term “endogenous,” within the context of the disclosure refers toany polynucleotide, polypeptide or protein sequence which is a naturalpart of a cell and is occurring at its natural location in the cellchromosome and of which the control of expression has not been alteredcompared to the natural control mechanism acting on its expression. Theterm “exogenous” refers to any polynucleotide, polypeptide or proteinsequence which originates from outside the cell under study and not anatural part of the cell or which is not occurring at its naturallocation in the cell chromosome or plasmid.

The term “heterologous” when used in reference to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme that is from a source orderived from a source other than the host organism species. In contrasta “homologous” polynucleotide, gene, nucleic acid, polypeptide, orenzyme is used herein to denote a polynucleotide, gene, nucleic acid,polypeptide, or enzyme that is derived from the host organism species.When referring to a gene regulatory sequence or to an auxiliary nucleicacid sequence used for maintaining or manipulating a gene sequence(e.g., a promoter, a 5′ untranslated region, 3′ untranslated region,poly A addition sequence, intron sequence, splice site, ribosome bindingsite, internal ribosome entry sequence, genome homology region,recombination site, etc.), “heterologous” means that the regulatorysequence or auxiliary sequence is not naturally associated with the genewith which the regulatory or auxiliary nucleic acid sequence isjuxtaposed in a construct, genome, chromosome, or episome. Thus, apromoter operably linked to a gene to which it is not operably linked toin its natural state (i.e., in the genome of a non-geneticallyengineered organism) is referred to herein as a “heterologous promoter,”even though the promoter may be derived from the same species (or, insome cases, the same organism) as the gene to which it is linked.

The term “modified activity” of a protein or an enzyme relates to achange in activity of the protein or the enzyme compared to the wildtype, i.e., natural, activity of the protein or enzyme. The modifiedactivity can either be an abolished, impaired, reduced or delayedactivity of the protein or enzyme compared to the wild type activity ofthe protein or the enzyme but can also be an accelerated or an enhancedactivity of the protein or the enzyme compared to the wild type activityof the protein or the enzyme. A modified activity of a protein or anenzyme is obtained by modified expression of the protein or enzyme or isobtained by expression of a modified, i.e., mutant form of the proteinor enzyme. A modified activity of an enzyme further relates to amodification in the apparent Michaelis constant Km and/or the apparentmaximal velocity (Vmax) of the enzyme.

The term “modified expression” of a gene relates to a change inexpression compared to the wild type expression of the gene in any phaseof the production process of the encoded protein. The modifiedexpression is either a lower or higher expression compared to the wildtype, wherein the term “higher expression” is also defined as“overexpression” of the gene in the case of an endogenous gene or“expression” in the case of a heterologous gene that is not present inthe wild type strain. Lower expression or reduced expression is obtainedby means of common well-known technologies for a skilled person (such asthe usage of siRNA, CrispR, CrispRi, riboswitches, recombineering,homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA,mutating genes, knocking-out genes, transposon mutagenesis, . . . )which are used to change the genes in such a way that they are less-able(i.e., statistically significantly ‘less-able’ compared to a functionalwild-type gene) or completely unable (such as knocked-out genes) toproduce functional final products. The term “riboswitch” as used hereinis defined to be part of the messenger RNA that folds into intricatestructures that block expression by interfering with translation.Binding of an effector molecule induces conformational change(s)permitting regulated expression post-transcriptionally. Next to changingthe gene of interest in such a way that lower expression is obtained asdescribed above, lower expression can also be obtained by changing thetranscription unit, the promoter, an untranslated region, the ribosomebinding site, the Shine Dalgarno sequence or the transcriptionterminator. Lower expression or reduced expression can, for instance, beobtained by mutating one or more base pairs in the promoter sequence orchanging the promoter sequence fully to a constitutive promoter with alower expression strength compared to the wild type or an induciblepromoter which result in regulated expression or a repressible promoterwhich results in regulated expression Overexpression or expression isobtained by means of common well-known technologies for a skilled person(such as the usage of artificial transcription factors, de novo designof a promoter sequence, ribosome engineering, introduction orre-introduction of an expression module at euchromatin, usage ofhigh-copy-number plasmids), wherein the gene is part of an “expressioncassette” which relates to any sequence in which a promoter sequence,untranslated region sequence (containing either a ribosome bindingsequence, Shine Dalgarno or Kozak sequence), a coding sequence andoptionally a transcription terminator is present, and leading to theexpression of a functional active protein. The expression is eitherconstitutive or regulated.

The term “constitutive expression” is defined as expression that is notregulated by transcription factors other than the sub units of RNApolymerase (e.g., the bacterial sigma factors like σ⁷⁰, σ⁵⁴, or relateds-factors and the yeast mitochondrial RNA polymerase specificity factorMTF1 that co-associate with the RNA polymerase core enzyme) undercertain growth conditions. Non-limiting examples of such transcriptionfactors are CRP, Lad, ArcA, Cra, Ica in E. coli, or, Aft2p, Crz1p, Skn7in Saccharomyces cerevisiae, or, DeoR, GntR, Fur in B. subtilis. Thesetranscription factors bind on a specific sequence and may block orenhance expression in certain growth conditions. The RNA polymerase isthe catalytic machinery for the synthesis of RNA from a DNA template.RNA polymerase binds a specific sequence to initiate transcription, forinstance, via a sigma factor in prokaryotic hosts or via MTF 1 inyeasts. Constitutive expression offers a constant level of expressionwith no need for induction or repression.

The term “regulated expression” is defined as expression that isregulated by transcription factors other than the subunits of RNApolymerase (e.g., bacterial sigma factors) under certain growthconditions. Examples of such transcription factors are described above.Commonly expression regulation is obtained by means of an inducer orrepressor, such as but not limited to IPTG, arabinose, rhamnose, fucose,allo-lactose or pH shifts, or temperature shifts or carbon depletion oracceptors or the produced product or chemical repression.

The term “expression by a natural inducer” is defined as a facultativeor regulatory expression of a gene that is only expressed upon a certainnatural condition of the host (e.g., organism being in labour, or duringlactation), as a response to an environmental change (e.g., includingbut not limited to hormone, heat, cold, pH shifts, light, oxidative orosmotic stress/signalling), or dependent on the position of thedevelopmental stage or the cell cycle of the host cell including but notlimited to apoptosis and autophagy.

The term “inducible expression upon chemical treatment” is defined as afacultative or regulatory expression of a gene that is only expressedupon treatment with a chemical inducer or repressor, wherein the inducerand repressor comprise but are not limited to an alcohol (e.g., ethanol,methanol), a carbohydrate (e.g., glucose, galactose, glycerol, lactose,arabinose, rhamnose, fucose, allo-lactose), metal ions (e.g., aluminum,copper, zinc), nitrogen, phosphates, IPTG, acetate, formate, xylene.

The term “control sequences” refers to sequences recognized by the cellstranscriptional and translational systems, allowing transcription andtranslation of a polynucleotide sequence to a polypeptide. Such DNAsequences are thus necessary for the expression of an operably linkedcoding sequence in a particular cell or organism. Such control sequencescan be, but are not limited to, promoter sequences, ribosome bindingsequences, Shine Dalgarno sequences, Kozak sequences, transcriptionterminator sequences. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers. DNA for apresequence or secretory leader may be operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. The control sequences can furthermore becontrolled with external chemicals, such as, but not limited to, IPTG,arabinose, lactose, allo-lactose, rhamnose or fucose via an induciblepromoter or via a genetic circuit that either induces or represses thetranscription or translation of the polynucleotide to a polypeptide.

Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.

The term “wild type” refers to the commonly known genetic orphenotypical situation as it occurs in nature.

The term “modified expression of a protein” as used herein refers to i)higher expression or overexpression of an endogenous protein, ii)expression of a heterologous protein or iii) expression and/oroverexpression of a variant protein that has a higher activity comparedto the wild-type (i.e., native) protein.

As used herein, the term “mammary cell(s)” generally refers to mammaryepithelial cell(s), mammary-epithelial luminal cell(s), or mammalianepithelial alveolar cell(s), or any combination thereof. As used herein,the term “mammary-like cell(s)” generally refers to cell(s) having aphenotype/genotype similar (or substantially similar) to natural mammarycell(s) but is/are derived from non-mammary cell source(s). Suchmammary-like cell(s) may be engineered to remove at least one undesiredgenetic component and/or to include at least one predetermined geneticconstruct that is typical of a mammary cell. Non-limiting examples ofmammary-like cell(s) may include mammary epithelial-like cell(s),mammary epithelial luminal-like cell(s), non-mammary cell(s) thatexhibits one or more characteristics of a cell of a mammary celllineage, or any combination thereof. Further non-limiting examples ofmammary-like cell(s) may include cell(s) having a phenotype similar (orsubstantially similar) to natural mammary cell(s), or more particularlya phenotype similar (or substantially similar) to natural mammaryepithelial cell(s). A cell with a phenotype or that exhibits at leastone characteristic similar to (or substantially similar to) a naturalmammary cell or a mammary epithelial cell may comprise a cell (e.g.,derived from a mammary cell lineage or a non-mammary cell lineage) thatexhibits either naturally, or has been engineered to, be capable ofexpressing at least one milk component.

As used herein, the term “non-mammary cell(s)” may generally include anycell of non-mammary lineage. In the context of the disclosure, anon-mammary cell can be any mammalian cell capable of being engineeredto express at least one milk component. Non-limiting examples of suchnon-mammary cell(s) include hepatocyte(s), blood cell(s), kidneycell(s), cord blood cell(s), epithelial cell(s), epidermal cell(s),myocyte(s), fibroblast(s), mesenchymal cell(s), or any combinationthereof. In some instances, molecular biology and genome editingtechniques can be engineered to eliminate, silence, or attenuate myriadgenes simultaneously.

Throughout the disclosure, unless explicitly stated otherwise, theexpressions “capable of . . . <verb>” and “capable to . . . <verb>” arepreferably replaced with the active voice of the verb and vice versa.For example, the expression “capable of expressing” is preferablyreplaced with “expresses” and vice versa, i.e., “expresses” ispreferably replaced with “capable of expressing.”

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to thepersons skilled in the art.

The term “derivative” of a polypeptide, as used herein, is a polypeptidewhich may contain deletions, additions or substitutions of amino acidresidues within the amino acid sequence of the polypeptide, but whichresult in a silent change, thus producing a functionally equivalentpolypeptide. Amino acid substitutions may be made based on similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; planar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Within the contextof this disclosure, a derivative polypeptide as used herein, refers to apolypeptide capable of exhibiting a substantially similar in vitroand/or in vivo activity as the original polypeptide as judged by any ofa number of criteria, including but not limited to enzymatic activity,and which may be differentially modified during or after translation.Furthermore, non-classical amino acids or chemical amino acid analoguescan be introduced as a substitution or addition into the originalpolypeptide sequence.

In some embodiments, the disclosure contemplates making functionalvariants by modifying the structure of an enzyme as used in thedisclosure. Variants can be produced by amino acid substitution,deletion, addition, or combinations thereof. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of a polypeptide of the disclosure results in afunctional homolog can be readily determined by assessing the ability ofthe variant polypeptide to produce a response in cells in a fashionsimilar to the wild-type polypeptide.

The term “functional homolog” as used herein describes those moleculesthat have sequence similarity (in other words, homology) and also shareat least one functional characteristic such as a biochemical activity(Altenhoff et al., PLoS Comput. Biol. 8 (2012) e1002514). Functionalhomologs will typically give rise to the same characteristics to asimilar, but not necessarily the same, degree. Functionally homologousproteins give the same characteristics where the quantitativemeasurement produced by one homolog is at least 10 percent of the other;more typically, at least 20 percent, between about 30 percent and about40 percent; for example, between about 50 percent and about 60 percent;between about 70 percent and about 80 percent; or between about 90percent and about 95 percent; between about 98 percent and about 100percent, or greater than 100 percent of that produced by the originalmolecule. Thus, where the molecule has enzymatic activity the functionalhomolog will have the above-recited percent enzymatic activitiescompared to the original enzyme. Where the molecule is a DNA-bindingmolecule (e.g., a polypeptide) the homolog will have the above-recitedpercentage of binding affinity as measured by weight of bound moleculecompared to the original molecule.

A functional homolog and the reference polypeptide may be naturallyoccurring polypeptides, and the sequence similarity may be due toconvergent or divergent evolutionary events. Functional homologs aresometimes referred to as orthologs, where “ortholog,” refers to ahomologous gene or protein that is the functional equivalent of thereferenced gene or protein in another species.

Orthologous genes are homologous genes in different species thatoriginate by vertical descent from a single gene of the last commonancestor, wherein the gene and its main function are conserved. Ahomologous gene is a gene inherited in two species by a common ancestor.

The term “ortholog” when used in reference to an amino acid ornucleotide/nucleic acid sequence from a given species refers to the sameamino acid or nucleotide/nucleic acid sequence from a different species.It should be understood that two sequences are orthologs of each otherwhen they are derived from a common ancestor sequence via linear descentand/or are otherwise closely related in terms of both their sequence andtheir biological function. Orthologs will usually have a high degree ofsequence identity but may not (and often will not) share 100% sequenceidentity.

Paralogous genes are homologous genes that originate by a geneduplication event. Paralogous genes often belong to the same species,but this is not necessary. Paralogs can be split into in-paralogs(paralogous pairs that arose after a speciation event) and out-paralogs(paralogous pairs that arose before a speciation event). Between speciesout-paralogs are pairs of paralogs that exist between two organisms dueto duplication before speciation. Within species out-paralogs are pairsof paralogs that exist in the same organism, but whose duplication eventhappened after speciation. Paralogs typically have the same or similarfunction.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofthe polypeptide of interest like e.g., a biomass-modulating polypeptide,a glycosyltransferase, a protein involved in nucleotide-activated sugarsynthesis or a membrane transporter protein. Sequence analysis caninvolve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundantdatabases using amino acid sequence of a biomass-modulating polypeptide,a glycosyltransferase, a protein involved in nucleotide-activated sugarsynthesis or a membrane transporter protein, respectively, as thereference sequence. Amino acid sequence is, in some instances, deducedfrom the nucleotide sequence. Typically, those polypeptides in thedatabase that have greater than 40 percent sequence identity arecandidates for further evaluation for suitability as abiomass-modulating polypeptide, a glycosyltransferase, a proteininvolved in nucleotide-activated sugar synthesis or a membranetransporter protein, respectively. Amino acid sequence similarity allowsfor conservative amino acid substitutions, such as substitution of onehydrophobic residue for another or substitution of one polar residue foranother or substitution of one acidic amino acid for another orsubstitution of one basic amino acid for another etc. Preferably, byconservative substitutions is intended combinations such as glycine byalanine and vice versa; valine, isoleucine and leucine by methionine andvice versa; aspartate by glutamate and vice versa; asparagine byglutamine and vice versa; serine by threonine and vice versa; lysine byarginine and vice versa; cysteine by methionine and vice versa; andphenylalanine and tyrosine by tryptophan and vice versa. If desired,manual inspection of such candidates can be carried out in order tonarrow the number of candidates to be further evaluated. Manualinspection can be performed by selecting those candidates that appear tohave domains present in productivity-modulating polypeptides, e.g.,conserved functional domains.

“Fragment,” with respect to a polynucleotide, refers to a clone or anypart of a polynucleotide molecule, particularly a part of apolynucleotide that retains a usable, functional characteristic of thefull-length polynucleotide molecule. Useful fragments includeoligonucleotides and polynucleotides that may be used in hybridizationor amplification technologies or in the regulation of replication,transcription or translation. A “polynucleotide fragment” refers to anysubsequence of a polynucleotide SEQ ID NO (or Genbank NO.), typically,comprising or consisting of at least about 9, 10, 11, 12 consecutivenucleotides from the polynucleotide SEQ ID NO (or Genbank NO.), forexample, at least about 30 nucleotides or at least about 50 nucleotidesof any of the polynucleotide sequences provided herein. Exemplaryfragments can additionally or alternatively include fragments thatcomprise, consist essentially of, or consist of a region that encodes aconserved family domain of a polypeptide. Exemplary fragments canadditionally or alternatively include fragments that comprise aconserved domain of a polypeptide. As such, a fragment of apolynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotidesequence which comprises or consists of the polynucleotide SEQ ID NO (orGenbank NO.) wherein no more than 200, 150, 100, 50 or 25 consecutivenucleotides are missing, preferably no more than 50 consecutivenucleotides are missing, and which retains a usable, functionalcharacteristic (e.g., activity) of the full-length polynucleotidemolecule which can be assessed by the skilled person through routineexperimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO(or Genbank NO.) preferably means a nucleotide sequence which comprisesor consists of an amount of consecutive nucleotides from thepolynucleotide SEQ ID NO (or Genbank NO.) and wherein the amount ofconsecutive nucleotides is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%,82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%,92.0%, 93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%,98.5%, 99.0%, 99.5%, 100%, preferably at least 80.0%, more preferably atleast 87.0%, even more preferably at least 90%, even more preferably atleast 95.0%, most preferably at least 97.0%, of the full-length of thepolynucleotide SEQ ID NO (or Genbank NO.) and retains a usable,functional characteristic (e.g., activity) of the full-lengthpolynucleotide molecule. As such, a fragment of a polynucleotide SEQ IDNO (or Genbank NO.) preferably means a nucleotide sequence whichcomprises or consists of the polynucleotide SEQ ID NO (or Genbank NO.),wherein an amount of consecutive nucleotides is missing and wherein theamount is no more than 50.0%, 40.0%, 30.0% of the full-length of thepolynucleotide SEQ ID NO (or Genbank NO.), preferably no more than20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%,3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably no more than 15.0%,even more preferably no more than 10.0%, even more preferably no morethan 5.0%, most preferably no more than 2.5%, of the full-length of thepolynucleotide SEQ ID NO (or Genbank NO.) and wherein the fragmentretains a usable, functional characteristic (e.g., activity) of thefull-length polynucleotide molecule which can be routinely assessed bythe skilled person.

Throughout the disclosure, the sequence of a polynucleotide can berepresented by a SEQ ID NO or alternatively by a GenBank NO. Therefore,the terms “polynucleotide SEQ ID NO” and “polynucleotide GenBank NO.”can be interchangeably used, unless explicitly stated otherwise.Fragments may additionally or alternatively include subsequences ofpolypeptides and protein molecules, or a subsequence of the polypeptide.In some cases, the fragment or domain is a subsequence of thepolypeptide which performs at least one biological function of theintact polypeptide in substantially the same manner, preferably to asimilar extent, as does the intact polypeptide. A “subsequence of thepolypeptide” as defined herein refers to a sequence of contiguous aminoacid residues derived from the polypeptide. For example, a polypeptidefragment can comprise a recognizable structural motif or functionaldomain such as a DNA-binding site or domain that binds to a DNA promoterregion, an activation domain, or a domain for protein-proteininteractions, and may initiate transcription. Fragments can vary in sizefrom as few as 3 amino acid residues to the full length of the intactpolypeptide, for example, at least about 20 amino acid residues inlength, for example, at least about 30 amino acid residues in length. Assuch, a fragment of a polypeptide SEQ ID NO (or UniProt ID or GenbankNO.) preferably means a polypeptide sequence which comprises or consistsof the polypeptide SEQ ID NO (or UniProt ID or Genbank NO.) wherein nomore than 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residuesare missing, preferably no more than 40 consecutive amino acid residuesare missing, and performs at least one biological function of the intactpolypeptide in substantially the same manner, preferably to a similar orgreater extent, as does the intact polypeptide which can be routinelyassessed by the skilled person. Alternatively, a fragment of apolypeptide SEQ ID NO (or UniProt ID or Genbank NO.) preferably means apolypeptide sequence which comprises or consists of an amount ofconsecutive amino acid residues from the polypeptide SEQ ID NO (orUniProt ID or Genbank NO.) and wherein the amount of consecutive aminoacid residues is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%,83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%,93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%,99.0%, 99.5%, 100%, preferably at least 80.0%, more preferably at least87.0%, even more preferably at least 90.0%, even more preferably atleast 95.0%, most preferably at least 97.0% of the full-length of thepolypeptide SEQ ID NO (or UniProt ID or Genbank NO.) and which performsat least one biological function of the intact polypeptide insubstantially the same manner, preferably to a similar or greaterextent, as does the intact polypeptide which can be routinely assessedby the skilled person. As such, a fragment of a polypeptide SEQ ID NO(or UniProt ID or Genbank NO.) preferably means a polypeptide sequencewhich comprises or consists of the polypeptide SEQ ID NO (or UniProt IDor Genbank NO.), wherein an amount of consecutive amino acid residues ismissing and wherein the amount is no more than 50.0%, 40.0%, 30.0% ofthe full-length of the polypeptide SEQ ID NO (or UniProt ID or GenbankNO.), preferably no more than 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%,6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, morepreferably no more than 15.0%, even more preferably no more than 10.0%,even more preferably no more than 5.0%, most preferably no more than2.5%, of the full-length of the polypeptide SEQ ID NO(or UniProt ID orGenbank NO.) and which performs at least one biological function of theintact polypeptide in substantially the same manner, preferably to asimilar or greater extent, as does the intact polypeptide which can beroutinely assessed by the skilled person.

Throughout the disclosure, the sequence of a polypeptide can berepresented by a SEQ ID NO or alternatively by a UniProt ID or GenBankNo. Therefore, the terms “polypeptide SEQ ID NO” and “polypeptideUniprot ID” and “polypeptide GenBank No.” can be interchangeably used,unless explicitly stated otherwise.

Preferentially a fragment of a polypeptide is a functional fragment thathas at least one property or activity of the polypeptide from which itis derived, preferably to a similar or greater extent. A functionalfragment can, for example, include a functional domain or conserveddomain of a polypeptide. It is understood that a polypeptide or afragment thereof may have conservative amino acid substitutions whichhave substantially no effect on the polypeptide's activity. Byconservative substitutions is intended substitutions of one hydrophobicamino acid for another or substitution of one polar amino acid foranother or substitution of one acidic amino acid for another orsubstitution of one basic amino acid for another etc. Preferably, byconservative substitutions is intended combinations such as glycine byalanine and vice versa; valine, isoleucine and leucine by methionine andvice versa; aspartate by glutamate and vice versa; asparagine byglutamine and vice versa; serine by threonine and vice versa; lysine byarginine and vice versa; cysteine by methionine and vice versa; andphenylalanine and tyrosine by tryptophan and vice versa. A domain can becharacterized, for example, by a Pfam (El-Gebali et al., Nucleic AcidsRes. 47 (2019) D427-D432) or Conserved Domain Database (CDD)(www.ncbi.nlm.nih.gov/cdd) (Lu et al., Nucleic Acids Res. 48 (2020)D265-D268) designation. The content of each database is fixed at eachrelease and is not to be changed. When the content of a specificdatabase is changed, this specific database receives a new releaseversion with a new release date. All release versions for each databasewith their corresponding release dates and specific content as annotatedat these specific release dates are available and known to those skilledin the art. The PFAM database (pfam.xfam.org/) used herein was Pfamversion 33.1 released on Jun. 11, 2020. Protein sequence information andfunctional information can be provided by a comprehensive resource forprotein sequence and annotation data like e.g., the Universal ProteinResource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1),D480-D489). UniProt comprises the expertly and richly curated proteindatabase called the UniProt Knowledgebase (UniProtKB), together with theUniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc).The UniProt identifiers (UniProt ID) are unique for each protein presentin the database. UniProt IDs as used herein are the UniProt IDs in theUniProt database version of 5 May 2021. Proteins that do not have anUniProt ID are referred herein using the respective GenBank Accessionnumber (GenBank No.) as present in the NIH genetic sequence database(www.ncbi.nlm.nih.gov/genbank/) (Nucleic Acids Res. 2013, 41(D1),D36-D42) version of 5 May 2021.

In the disclosure, polypeptide sequence stretches are being used torefer to fragments of the N-acetylglucosamineb-1,3-galactosyltransferases and the N-acetylglucosamineb-1,4-galactosyltransferases used in the disclosure which are common tothose N-acetylglucosamine b-1,3-galactosyltransferases andN-acetylglucosamine b-1,4-galactosyltransferases. Such polypeptidestretches are written in the form of a sequence of amino acids inone-letter code. In case an amino acid at a specific place in suchpolypeptide stretch can be several amino acids, that specific place willhave amino acid code X. Unless otherwise mentioned herein, the letter“X” refers to any amino acid possible. The term (Xn) refers to a stretchof a protein sequence consisting of a number n of the amino acid residueX wherein each of the X is any amino acid possible and wherein n is 2,3, 4 or more. The term (Xm) refers to a stretch of a protein sequenceconsisting of a number m of the amino acid residue X wherein each of theX is any amino acid possible and wherein m is 2, 3, 4 or more. The term(Xp) refers to a stretch of a protein sequence consisting of a number pof the amino acid residue X wherein each of the X is any amino acidpossible and wherein p is 2, 3, 4 or more. The term “[X, no A, G or S]”refers to any amino acid excluding the amino acid residues alanine (A),glycine (G) or serine (S). The term “[X, no F, H, W or Y]” refers to anyamino acid excluding the amino acid residues phenylalanine (F),histidine (H), tryptophan (W) and tyrosine (Y).

The terms “nucleotide-sugar” or “activated sugar” as used herein referto activated forms of monosaccharides. Examples of activatedmonosaccharides include but are not limited to UDP-galactose (UDP-Gal),UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine(UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose(GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, or UDP-xylose. Nucleotide-sugars act as glycosyl donors inglycosylation reactions. Those reactions are catalyzed by a group ofenzymes called glycosyltransferases.

The terms “N-acetylglucosamine b-1,3-galactosyltransferase,”“N-acetylglucosamine beta-1,3-galactosyltransferase,”“N-acetylglucosamine beta 1,3 galactosyltransferase,”“N-acetylglucosamine β-1,3-galactosyltransferase,” “N-acetylglucosamineβ1,3 galactosyltransferase” as used in the disclosure, are usedinterchangeably and refer to a galactosyltransferase that catalyses thetransfer of galactose from the donor UDP-galactose, to the acceptorN-acetylglucosamine in an beta-1,3 glycosidic linkage. A polynucleotideencoding an “N-acetylglucosamine b-1,3-galactosyltransferase” or any ofthe above terms, refers to a polynucleotide encoding suchglycosyltransferase that catalyses the transfer of galactose from thedonor UDP-galactose, to the acceptor N-acetylglucosamine in an beta-1,3glycosidic linkage.

The terms “N-acetylglucosamine b-1,4-galactosyltransferase,”“N-acetylglucosamine beta-1,4-galactosyltransferase,”“N-acetylglucosamine beta 1,4 galactosyltransferase,”“N-acetylglucosamine β-1,4-galactosyltransferase,” “N-acetylglucosamineβ1,4 galactosyltransferase” as used in the disclosure, are usedinterchangeably and refer to a galactosyltransferase that catalyses thetransfer of galactose from the donor UDP-galactose, to the acceptorN-acetylglucosamine in an beta-1,4 glycosidic linkage. A polynucleotideencoding an “N-acetylglucosamine b-1,4-galactosyltransferase” or any ofthe above terms, refers to a polynucleotide encoding suchglycosyltransferase that catalyses the transfer of galactose from thedonor UDP-galactose, to the acceptor N-acetylglucosamine in an beta-1,4glycosidic linkage.

The terms “glucosamine 6-phosphate N-acetyltransferase,”“glucosamine-phosphate N-acetyltransferase,” “GNA,” “GNA1,”“glucosamine-6P N-acetyltransferase,” “GlcN6P N-acetyltransferase” asused in the disclosure, are used interchangeably and refer to an enzymethat catalyses the transfer of an acetyl group from acetyl-CoA to theprimary amine in glucosamine-6-phosphate, generatingN-acetyl-D-glucosamine-6-phosphate, the latter also known as GlcNAc-6P.A polynucleotide encoding a “glucosamine 6-phosphateN-acetyltransferase” or any of the above terms, refers to apolynucleotide encoding such an enzyme that catalyses the transfer of anacetyl group from acetyl-CoA to the primary amine inglucosamine-6-phosphate, generating N-acetyl-D-glucosamine-6-phosphate.

The terms “fructose-6-phosphate aminotransferase,”“glutamine-fructose-6-phosphate-aminotransferase,”“glutamine-fructose-6-phosphate aminotransferase,”“L-glutamine-D-fructose-6-phosphate aminotransferase,” “glmS,” “glms,”“glmS*54” as used in the disclosure, are used interchangeably and referto an enzyme that catalyzes the conversion of fructose-6-phosphate intoglucosamine-6-phosphate using glutamine as a nitrogen source. Apolynucleotide encoding a “fructose-6-phosphate aminotransferase” or anyof the above terms, refers to a polynucleotide encoding such an enzymethat catalyzes the conversion of fructose-6-phosphate intoglucosamine-6-phosphate using glutamine as a nitrogen source.

The term “bioproduct” as used herein refers to a di- or oligosaccharidehaving an N-acetylglucosamine unit at the reducing end that issynthesized in a biological manner, i.e., via microbial synthesis,cellular synthesis.

The term “disaccharide” as used herein refers to a saccharide composedof two monosaccharide units. “Oligosaccharide” as the term is usedherein and as generally understood in the state of the art, refers to asaccharide polymer containing a small number, typically three to twenty,of simple sugars, i.e., monosaccharides. The monosaccharides as usedherein are reducing sugars. The disaccharides and oligosaccharides canbe reducing or non-reducing sugars and have a reducing and anon-reducing end. A reducing sugar is any sugar that is capable ofreducing another compound and is oxidized itself, that is, the carbonylcarbon of the sugar is oxidized to a carboxyl group. The term “reducingend of a saccharide” as used in the disclosure, refers to the freeanomeric carbon that is available in the saccharide to reduce anothercompound.

The term “monosaccharide” as used herein refers to a sugar that is notdecomposable into simpler sugars by hydrolysis, is classed either analdose or ketose, and contains one or more hydroxyl groups per molecule.Monosaccharides are saccharides containing only one simple sugar.Examples of monosaccharides comprise Hexose, D-Glucopyranose,D-Galactofuranose, D-Galactopyranose, L-Galactopyranose,D-Mannopyranose, D-Allopyranose, L-Altropyranose, D-Gulopyranose,L-Idopyranose, D-Talopyranose, D-Ribofuranose, D-Ribopyranose,D-Arabinofuranose, D-Arabinopyranose, L-Arabinofuranose,L-Arabinopyranose, D-Xylopyranose, D-Lyxopyranose, D-Erythrofuranose,D-Threofuranose, Heptose, L-glycero-D-manno-Heptopyranose (LDmanHep),D-glycero-D-manno-Heptopyranose (DDmanHep), 6-Deoxy-L-altropyranose,6-Deoxy-D-gulopyranose, 6-Deoxy-D-talopyranose,6-Deoxy-D-galactopyranose, 6-Deoxy-L-galactopyranose,6-Deoxy-D-mannopyranose, 6-Deoxy-L-mannopyranose,6-Deoxy-D-glucopyranose, 2-Deoxy-D-arabino-hexose,2-Deoxy-D-erythro-pentose, 2,6-Dideoxy-D-arabino-hexopyranose,3,6-Dideoxy-D-arabino-hexopyranose, 3,6-Dideoxy-L-arabino-hexopyranose,3,6-Dideoxy-D-xylo-hexopyranose, 3,6-Dideoxy-D-ribo-hexopyranose,2,6-Dideoxy-D-ribo-hexopyranose, 3,6-Dideoxy-L-xylo-hexopyranose,2-Amino-2-deoxy-D-glucopyranose, 2-Amino-2-deoxy-D-galactopyranose,2-Amino-2-deoxy-D-mannopyranose, 2-Amino-2-deoxy-D-allopyranose,2-Amino-2-deoxy-L-altropyranose, 2-Amino-2-deoxy-D-gulopyranose,2-Amino-2-deoxy-L-idopyranose, 2-Amino-2-deoxy-D-talopyranose,2-Acetamido-2-deoxy-D-glucopyranose,2-Acetamido-2-deoxy-D-galactopyranose,2-Acetamido-2-deoxy-D-mannopyranose, 2-Acetamido-2-deoxy-D-allopyranose,2-Acetamido-2-deoxy-L-altropyranose, 2-Acetamido-2-deoxy-D-gulopyranose,2-Acetamido-2-deoxy-L-idopyranose, 2-Acetamido-2-deoxy-D-talopyranose,2-Acetamido-2,6-dideoxy-D-galactopyranose,2-Acetamido-2,6-dideoxy-L-galactopyranose,2-Acetamido-2,6-dideoxy-L-mannopyranose,2-Acetamido-2,6-dideoxy-D-glucopyranose,2-Acetamido-2,6-dideoxy-L-altropyranose,2-Acetamido-2,6-dideoxy-D-talopyranose, D-Glucopyranuronic acid,D-Galactopyranuronic acid, D-Mannopyranuronic acid, D-Allopyranuronicacid, L-Altropyranuronic acid, D-Gulopyranuronic acid, L-Gulopyranuronicacid, L-Idopyranuronic acid, D-Talopyranuronic acid, sialic acid,5-Amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid,5-Acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid,5-Glycolylamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid,Erythritol, Arabinitol, Xylitol, Ribitol, Glucitol, Galactitol,Mannitol, D-ribo-Hex-2-ulopyranose, D-arabino-Hex-2-ulofuranose(D-fructofuranose), D-arabino-Hex-2-ulopyranose,L-xylo-Hex-2-ulopyranose, D-lyxo-Hex-2-ulopyranose,D-threo-Pent-2-ulopyranose, D-altro-Hept-2-ulopyranose,3-C-(Hydroxymethyl)-D-erythofuranose,2,4,6-Trideoxy-2,4-diamino-D-glucopyranose,6-Deoxy-3-O-methyl-D-glucose, 3-O-Methyl-D-rhamnose,2,6-Dideoxy-3-methyl-D-ribo-hexose,2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose,2-Acetamido-3-O—[(R)-carboxyethyl]-2-deoxy-D-glucopyranose,2-Glycolylamido-3-O—[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose,3-Deoxy-D-lyxo-hept-2-ulopyranosaric acid,3-Deoxy-D-manno-oct-2-ulopyranosonic acid,3-Deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid,5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulopyranosonicacid,5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulopyranosonicacid,5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulopyranosonicacid,5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulopyranosonicacid, glucose, galactose, N-acetylglucosamine, glucosamine, mannose,xylose, N-acetylmannosamine, N-acetylneuraminic acid,N-glycolylneuraminic acid, N-acetylgalactosamine, galactosamine, fucose,rhamnose, glucuronic acid, gluconic acid, fructose and polyols.

With the term polyol is meant an alcohol containing multiple hydroxylgroups. For example, glycerol, sorbitol, or mannitol.

The term “disaccharide having an N-acetylglucosamine unit at thereducing end” includes but is not limited to the productsGal-b1,3-GlcNAc or Gal-b1,4-GlcNAc, wherein galactose is linked to anN-acetylglucosamine in a beta-1,3-linkage or a beta-1,4-linkage,respectively, and wherein N-acetylglucosamine is positioned at thereducing end of the disaccharide.

The terms “Gal-b1,3-GlcNAc,” “Gal-beta-1,3-GlcNAc,” “Gal-β1,3-GlcNAc,”“Galb1,3GlcNAc,” “Galβ1,3GlcNAc,” “lacto-N-biose,” “LNB,” “LacNAc typeI,” “type 1 LacNAc,” “LacNAc (I)” are used interchangeably and refer toa disaccharide wherein galactose is linked to an N-acetylglucosamine ina beta-1,3-linkage, and wherein N-acetylglucosamine is positioned at thereducing end of the disaccharide.

The terms “Gal-b1,4-GlcNAc,” “Gal-beta-1,4-GlcNAc,” “Gal-β1,4-GlcNAc,”“Galb1,4GlcNAc,” “Galβ1,4GlcNAc,” “N-acetyllactosamine,” “LacNAc,”“LacNAc type II,” “type 2 LacNAc,” “LacNAc (II)” are usedinterchangeably and refer to a disaccharide wherein galactose is linkedto an N-acetylglucosamine in a beta-1,4-linkage, and whereinN-acetylglucosamine is positioned at the reducing end of thedisaccharide.

The term “oligosaccharide having an N-acetylglucosamine unit at thereducing end” as used in the disclosure refers to an oligosaccharidebuilt of three to twenty monosaccharide units, wherein anN-acetylglucosamine is present at the reducing end of theoligosaccharide. The oligosaccharide as used in the disclosure can be alinear structure or can include branches. The linkage (e.g., glycosidiclinkage, galactosidic linkage, glucosidic linkage, etc.) between twosugar units can be expressed, for example, as 1,4, 1->4, or (1-4), usedinterchangeably herein. For example, the terms “Gal-b1,4-Glc,”“β-Gal-(1->4)-Glc,” “Galbeta1-4-Glc” and “Gal-b(1-4)-Glc” have the samemeaning, i.e., a beta-glycosidic bond links carbon-1 of galactose (Gal)with the carbon-4 of glucose (Glc). Each monosaccharide can be in thecyclic form (e.g., pyranose of furanose form). Linkages between theindividual monosaccharide units may include alpha 1->2, alpha 1->3,alpha 1->4, alpha 1->6, alpha 2->1, alpha 2->3, alpha 2->4, alpha 2->6,beta 1->2, beta 1->3, beta 1->4, beta 1->6, beta 2->1, beta 2->3, beta2->4, and beta 2->6. An oligosaccharide can contain both alpha- andbeta-glycosidic bonds or can contain only beta-glycosidic bonds. Anoligosaccharide as used in the disclosure can be defined according tothe formula 1:

wherein the oligosaccharide is composed of the following monosaccharidesbound in either alpha or beta glycosidic bonds, and wherein B isN-acetylglucosamine, and wherein A, V, W, X, Y, and/or Z are absent, orare a galactose, glucose, fucose, mannose, xylose, glucuronic acid,galacturonic acid, iduronic acid, N-acetylneuraminic acid,N-glycolylneuraminic acid, glucosamine, N-acetylgalactosamine,N-acetylmannosamine, N-acetylglucosamine and/or an oligosaccharidestructure as defined by the formula 2:

wherein B is N-acetylglucosamine, and wherein A, V, W, X, Y, and/or Zare absent, or are a galactose, glucose, fucose, mannose, xylose,glucuronic acid, galacturonic acid, iduronic acid, N-acetylneuraminicacid, N-glycolylneuraminic acid, glucosamine, N-acetylgalactosamine,N-acetylmannosamine or N-acetylglucosamine; and wherein, in formula 1and in formula 2, m is 3 with optionally v is 4, or m is 4 withoptionally v is 3, and wherein p is 3 and/or 4, and wherein w is 6, andwherein x is 3 and X is no monosaccharide if n>1 and p is 3, and whereiny is 4 and Y is no monosaccharide if n>1 and p is 4, and wherein z is 6,and wherein n ranges from 1 to 10.

As used herein, “mammalian milk oligosaccharide” (MMO) refers tooligosaccharides such as but not limited to lacto-N-triose II,3-fucosyllactose, 2′-fucosyllactose, 6-fucosyllactose,2′,3-difucosyllactose, 2′,2-difucosyllactose, 3,4-difucosyllactose,6′-sialyllactose, 3′-sialyllactose, 3,6-disialyllactose,6,6′-disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose,lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose,lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaoseIII, lacto-N-fucopentaose V, lacto-N-fucopentaose VI,sialyllacto-N-tetraose c, sialyllacto-N-tetraose b,sialyllacto-N-tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaoseII, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose,monofucosylmonosialyllacto-N-tetraose c, monofucosylpara-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomericfucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I,sialyllacto-N-hexaose, sialyllacto-N-neohexaose II,difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose,difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylatedchitosan, fucosylated oligosaccharides, neutral oligosaccharide and/orsialylated oligosaccharides. Mammalian milk oligosaccharides (MMOs)comprise oligosaccharides present in milk found in any phase duringlactation including colostrum milk from humans (i.e., human milkoligosaccharides or HMOs) and mammals including but not limited to cows(Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus),bactrian camels (Camelus bactrianus), horses (Equus ferus caballus),pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears(Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese blackbears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis),hooded seals (Cystophora cristata), Asian elephants (Elephas maximus),African elephant (Loxodonta africana), giant anteater (Myrmecophagatridactyla), common bottlenose dolphins (Tursiops truncates), northernminke whales (Balaenoptera acutorostrata), tammar wallabies (Macropuseugenii), red kangaroos (Macropus rufus), common brushtail possum(Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls(Dasyurus viverrinus), platypus (Ornithorhynchus anatinus). Human milkoligosaccharides (HMOs) are also known as human identical milkoligosaccharides which are chemically identical to the human milkoligosaccharides found in human breast milk but which arebiotechnologically-produced (e.g., using cell free systems or cells andorganisms comprising a bacterium, a fungus, a yeast, a plant, animal, orprotozoan cell, preferably genetically engineered cells and organisms).Human identical milk oligosaccharides are marketed under the name HiMO.

The term “purified” refers to material that is substantially oressentially free from components which interfere with the activity ofthe biological molecule. For cells, saccharides, nucleic acids, andpolypeptides, the term “purified” refers to material that issubstantially or essentially free from components which normallyaccompany the material as found in its native state. Typically, purifiedsaccharides, oligosaccharides, proteins or nucleic acids of thedisclosure are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% pure as measured by band intensity on a silverstained gel orother method for determining purity. Purity or homogeneity can beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein or nucleic acid sample,followed by visualization upon staining. For certain purposes highresolution will be needed and HPLC or a similar means for purificationutilized. For oligosaccharides, purity can be determined using methodssuch as but not limited to thin layer chromatography, gaschromatography, NMR, HPLC, capillary electrophoresis or massspectroscopy.

The terms “identical” or “percent identity” or “% identity” in thecontext of two or more nucleic acid or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, as measuredusing sequence comparison algorithms or by visual inspection. Forsequence comparison, one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are inputted into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. Percent identity may be calculated globally over thefull-length sequence of the reference sequence, resulting in a globalpercent identity score. Alternatively, percent identity may becalculated over a partial sequence of the reference sequence, resultingin a local percent identity score. Using the full-length of thereference sequence in a local sequence alignment results in a globalpercent identity score between the test and the reference sequence.Percent identity can be determined using different algorithms, forexample, BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3,403-410; Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389-402), theClustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), theMatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) orEMBOSS Needle.

The BLAST (Basic Local Alignment Search Tool)) method of alignment is analgorithm provided by the National Center for Biotechnology Information(NCBI) to compare sequences using default parameters. The programcompares nucleotide or protein sequences to sequence databases andcalculates the statistical significance. PSI-BLAST (Position-SpecificIterative Basic Local Alignment Search Tool) derives a position-specificscoring matrix (PSSM) or profile from the multiple sequence alignment ofsequences detected above a given score threshold using protein—proteinBLAST (BLASTp). The BLAST method can be used for pairwise or multiplesequence alignments. Pairwise Sequence Alignment is used to identifyregions of similarity that may indicate functional, structural and/orevolutionary relationships between two biological sequences (protein ornucleic acid). The web interface for BLAST is available at:blast.ncbi.nlm.nih.gov/Blast.cgi.

Clustal Omega (Clustal W) is a multiple sequence alignment program thatuses seeded guide trees and HMM profile-profile techniques to generatealignments between three or more sequences. It produces biologicallymeaningful multiple sequence alignments of divergent sequences. The webinterface for Clustal W is available atwww.ebi.ac.uk/Tools/msa/clustalo/. Default parameters for multiplesequence alignments and calculation of percent identity of proteinsequences using the Clustal W method are: enabling de-alignment of inputsequences: FALSE; enabling mbed-like clustering guide-tree: TRUE;enabling mbed-like clustering iteration: TRUE; Number of (combinedguide-tree/HMM) iterations: default(0); Max Guide Tree Iterations:default [−1]; Max HMM Iterations: default [−1]; order: aligned.

MatGAT (Matrix Global Alignment Tool) is a computer application thatgenerates similarity/identity matrices for DNA or protein sequenceswithout needing pre-alignment of the data. The program performs a seriesof pairwise alignments using the Myers and Miller global alignmentalgorithm, calculates similarity and identity, and then places theresults in a distance matrix. The user may specify which type ofalignment matrix (e.g., BLOSUM50, BLOSUM62, and PAM250) to employ withtheir protein sequence examination.

EMBOSS Needle (galaxy-iuc.github.io/emboss-5.0-docs/needle.html) usesthe Needleman-Wunsch global alignment algorithm to find the optimalalignment (including gaps) of two sequences when considering theirentire length. The optimal alignment is ensured by dynamic programmingmethods by exploring all possible alignments and choosing the best. TheNeedleman-Wunsch algorithm is a member of the class of algorithms thatcan calculate the best score and alignment in the order of mn steps,(where ‘n’ and ‘m’ are the lengths of the two sequences). The gap openpenalty (default 10.0) is the score taken away when a gap is created.The default value assumes you are using the EBLOSUM62 matrix for proteinsequences. The gap extension (default 0.5) penalty is added to thestandard gap penalty for each base or residue in the gap. This is howlong gaps are penalized.

As used herein, a polypeptide having an amino acid sequence having atleast 80% sequence identity to the full-length sequence of a referencepolypeptide sequence is to be understood as that the sequence has 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.50%, 92.00%,92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%,97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%,99.90%, 100% sequence identity to the full-length of the amino acidsequence of the reference polypeptide sequence. Throughout thedisclosure, unless explicitly specified otherwise, a polypeptide (or DNAsequence) comprising/consisting/having an amino acid sequence (ornucleotide sequence) having at least 80% sequence identity to thefull-length amino acid sequence (or nucleotide sequence) of a referencepolypeptide (or nucleotide sequence), usually indicated with a SEQ ID NOor UniProt ID or Genbank NO., preferably has at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably has at least85%, even more preferably has at least 90%, most preferably has at least95%, sequence identity to the full length reference sequence.

For the purposes of this disclosure, percent identity is determinedusing MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). Thefollowing default parameters for protein are employed: (1) Gap costExistence: 12 and Extension: 2; (2) The Matrix employed was BLOSUM65. Ina preferred embodiment, sequence identity is calculated based on thefull-length sequence of a given SEQ ID NO, i.e., the reference sequence,or a part thereof. Part thereof preferably means at least 50%, 60%, 70%,80%, 90% or 95% of the complete reference sequence.

The term “cultivation” refers to the culture medium wherein the cell iscultivated or fermented, the cell itself, and the di- and/oroligosaccharides with a GlcNAc at their reducing end that are producedby the cell of the disclosure in whole broth, i.e., inside(intracellularly) as well as outside (extracellularly) of the cell.

The term “membrane transporter proteins” as used herein refers toproteins that are part of or interact with the cell membrane and controlthe flow of molecules and information across the cell. The membraneproteins are thus involved in transport, be it import into or export outof the cell.

Such membrane transporter proteins can be porters,P—P-bond-hydrolysis-driven transporters, β-Barrel Porins, auxiliarytransport proteins, putative transport proteins andphosphotransfer-driven group translocators as defined by the TransporterClassification Database that is operated and curated by the Saier LabBioinformatics Group available via www.tcdb.org and providing afunctional and phylogenetic classification of membrane transportproteins This Transporter Classification Database details acomprehensive IUBMB approved classification system for membranetransporter proteins known as the Transporter Classification (TC)system. The TCDB classification searches as described here are definedbased on TCDB. org as released on 17 Jun. 2019.

Porters is the collective name of uniporters, symporters, andantiporters that utilize a carrier-mediated process (Saier et al.,Nucleic Acids Res. 44 (2016) D372-D379). They belong to theelectrochemical potential-driven transporters and are also known assecondary carrier-type facilitators. Membrane transporter proteins areincluded in this class when they utilize a carrier-mediated process tocatalyze uniport when a single species is transported either byfacilitated diffusion or in a membrane potential-dependent process ifthe solute is charged; antiport when two or more species are transportedin opposite directions in a tightly coupled process, not coupled to adirect form of energy other than chemiosmotic energy; and/or symportwhen two or more species are transported together in the same directionin a tightly coupled process, not coupled to a direct form of energyother than chemiosmotic energy, of secondary carriers (Forrest et al.,Biochim. Biophys. Acta 1807 (2011) 167-188). These systems are usuallystereospecific. Solute:solute countertransport is a characteristicfeature of secondary carriers. The dynamic association of porters andenzymes creates functional membrane transport metabolons that channelsubstrates typically obtained from the extracellular compartmentdirectly into their cellular metabolism (Moraes and Reithmeier, Biochim.Biophys. Acta 1818 (2012), 2687-2706). Solutes that are transported viathis porter system include but are not limited to cations, organicanions, inorganic anions, nucleosides, amino acids, polyols,phosphorylated glycolytic intermediates, osmolytes, siderophores.

Membrane transporter proteins are included in the class of P—P-bondhydrolysis-driven transporters if they hydrolyze the diphosphate bond ofinorganic pyrophosphate, ATP, or another nucleoside triphosphate, todrive the active uptake and/or extrusion of a solute or solutes (Saieret al., Nucleic Acids Res. 44 (2016) D372-D379). The membranetransporter protein may or may not be transiently phosphorylated, butthe substrate is not phosphorylated. Substrates that are transported viathe class of P—P-bond hydrolysis-driven transporters include but are notlimited to cations, heavy metals, beta-glucan, UDP-glucose,lipopolysaccharides, teichoic acid.

The β-Barrel porins membrane transporter proteins form transmembranepores that usually allow the energy independent passage of solutesacross a membrane. The transmembrane portions of these proteins consistexclusively of β-strands which form a β-barrel (Saier et al., NucleicAcids Res. 44 (2016) D372-D379). These porin-type proteins are found inthe outer membranes of Gram-negative bacteria, mitochondria, plastids,and possibly acid-fast Gram-positive bacteria. Solutes that aretransported via these β-Barrel porins include but are not limited tonucleosides, raffinose, glucose, beta-glucosides, oligosaccharides.

The auxiliary transport proteins are defined to be proteins thatfacilitate transport across one or more biological membranes but do notthemselves participate directly in transport. These membrane transporterproteins always function in conjunction with one or more establishedtransport systems such as but not limited to outer membrane factors(OMFs), polysaccharide (PST) porters, the ATP-binding cassette(ABC)-type transporters. They may provide a function connected withenergy coupling to transport, play a structural role in complexformation, serve a biogenic or stability function or function inregulation (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379).Examples of auxiliary transport proteins include but are not limited tothe polysaccharide copolymerase family involved in polysaccharidetransport, the membrane fusion protein family involved in bacteriocinand chemical toxin transport.

Putative transport protein comprises families which will either beclassified elsewhere when the transport function of a member becomesestablished or will be eliminated from the Transporter Classificationsystem if the proposed transport function is disproven. These familiesinclude a member or members for which a transport function has beensuggested, but evidence for such a function is not yet compelling (Saieret al., Nucleic Acids Res. 44 (2016) D372-D379). Examples of putativetransporters classified in this group under the TCDB system as releasedon 17 Jun. 2019 include but are not limited to copper transporters.

The phosphotransfer-driven group translocators are also known as thePEP-dependent phosphoryl transfer-driven group translocators of thebacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). Theproduct of the reaction, derived from extracellular sugar, is acytoplasmic sugar-phosphate. The enzymatic constituents, catalyzingsugar phosphorylation, are superimposed on the transport process in atightly coupled process. The PTS system is involved in many differentaspects comprising in regulation and chemotaxis, biofilm formation, andpathogenesis (Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93;Saier, J. Mol. Microbiol. Biotechnol. 25 (2015) 73-78). Membranetransporter protein families classified within thephosphotransfer-driven group translocators under the TCDB system asreleased on 17 Jun. 2019 include PTS systems linked to transport ofglucose-glucosides, fructose-mannitol,lactose-N,N′-diacetylchitobiose-beta-glucoside, glucitol, galactitol,mannose-fructose-sorbose and ascorbate.

The major facilitator superfamily (MFS) is a superfamily of membranetransporter proteins catalyzing uniport, solute:cation (H+, but seldomNa+) symport and/or solute:H+ or solute:solute antiport. Most are of400-600 amino acyl residues in length and possess either 12, 14, oroccasionally, 24 transmembrane α-helical spanners (TMSs) as defined bythe Transporter Classification Database operated by the Saier LabBioinformatics Group (www.tcdb.org).

“SET” or “Sugar Efflux Transporter” as used herein refers to membraneproteins of the SET family which are proteins with InterPRO domainIPR004750 and/or are proteins that belong to the eggNOGv4.5 familyENOG410XTE9. Identification of the InterPro domain can be done by usingthe online tool on www.ebi.ac.uk/interpro/or a standalone version ofInterProScan (www.ebi.ac.uk/interpro/download.html) using the defaultvalues. Identification of the orthology family in eggNOGv4.5 can be doneusing the online version or a standalone version of eggNOG-mapperv1(eggnogdb.embl.de/#/app/home).

The term “Siderophore” as used herein is referring to the secondarymetabolite of various microorganisms which are mainly ferric ionspecific chelators. These molecules have been classified as catecholate,hydroxamate, carboxylate and mixed types. Siderophores are in generalsynthesized by a nonribosomal peptide synthetase (NRPS) dependentpathway or an NRPS independent pathway (NIS). The most importantprecursor in NRPS-dependent siderophore biosynthetic pathway ischorismate. 2, 3-DHBA could be formed from chorismate by a three-stepreaction catalyzed by isochorismate synthase, isochorismatase, and 2,3-dihydroxybenzoate-2, 3-dehydrogenase. Siderophores can also be formedfrom salicylate which is formed from isochorismate by isochorismatepyruvate lyase. When ornithine is used as precursor for siderophores,biosynthesis depends on the hydroxylation of ornithine catalyzed byL-ornithine N5-monooxygenase. In the NIS pathway, an important step insiderophore biosynthesis is N(6)-hydroxylysine synthase.

A transporter is needed to export the siderophore outside the cell. Foursuperfamilies of membrane proteins are identified so far in thisprocess: the major facilitator superfamily (MFS); theMultidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily(MOP); the resistance, nodulation and cell division superfamily (RND);and the ABC superfamily. In general, the genes involved in siderophoreexport are clustered together with the siderophore biosynthesis genes.The term “siderophore exporter” as used herein refers to suchtransporters needed to export the siderophore outside of the cell.

The ATP-binding cassette (ABC) superfamily contains both uptake andefflux transport systems, and the members of these two groups generallycluster loosely together. ATP hydrolysis without protein phosphorylationenergizes transport. There are dozens of families within the ABCsuperfamily, and family generally correlates with substrate specificity.Members are classified according to class 3.A.1 as defined by theTransporter Classification Database operated by the Saier LabBioinformatics Group available via www.tcdb.org and providing afunctional and phylogenetic classification of membrane transporterproteins.

The term “enabled efflux” means to introduce the activity of transportof a solute over the cytoplasm membrane and/or the cell wall. Thetransport may be enabled by introducing and/or increasing the expressionof a transporter protein as described in the disclosure. The term“enhanced efflux” means to improve the activity of transport of a soluteover the cytoplasm membrane and/or the cell wall. Transport of a soluteover the cytoplasm membrane and/or cell wall may be enhanced byintroducing and/or increasing the expression of a membrane transporterprotein as described in the disclosure. “Expression” of a membranetransporter protein is defined as “overexpression” of the gene encodingthe membrane transporter protein in the case the gene is an endogenousgene or “expression” in the case the gene encoding the membranetransporter protein is a heterologous gene that is not present in thewild type strain or cell.

The term “precursor” as used herein refers to substances which are takenup and/or synthetized by the cell for the specific production of a di-and/or oligosaccharide like e.g., a di- or oligosaccharide with anN-acetylglucosamine unit at the reducing end. In this sense a precursorcan be an acceptor as defined herein, but can also be another substance,metabolite, which is first modified within the cell as part of thebiochemical synthesis route of the di- and/or oligosaccharide like e.g.,a di- or oligosaccharide with an N-acetylglucosamine unit at thereducing end. Examples of such precursors comprise the acceptors asdefined herein, and/or glucose, galactose, fructose, glycerol, sialicacid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone,glucosamine, mannosamine, N-acetyl-mannosamine, galactosamine,N-acetylgalactosamine, phosphorylated sugars like e.g., but not limitedto glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate,fructose-6-phosphate, fructose-1,6-bisphosphate, mannose-6-phosphate,mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate,dihydroxyacetone-phosphate, glucosamine-6-phosphate,N-acetyl-glucosamine-6-phosphate, N-acetylmannosamine-6-phosphate,N-acetylglucosamine-1-phosphate, N-acetyl-neuraminic acid-9-phosphateand/or nucleotide-activated sugars as defined herein like e.g.,UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid,GDP-mannose, GDP-4-dehydro-6-deoxy-α-D-mannose, GDP-fucose.

The term “acceptor” as used herein refers to a mono-, di- oroligosaccharide which can be modified by a glycosyltransferase. Examplesof such acceptors comprise glucose, galactose, fructose, glycerol,sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-biose(LNB), lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose(LNnT), N-acetyl-lactosamine (LacNAc), lacto-N-pentaose (LNP),lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose,lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose(LNnH), para lacto-N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH),lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, paralacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, isolacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novolacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novolacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose,novo lacto-N-decaose, lacto-N-neodecaose, galactosyllactose, a lactoseextended with 1, 2, 3, 4, 5, or a multiple of N-acetyllactosamine unitsand/or 1, 2, 3, 4, 5, or a multiple of, Lacto-N-biose units, andoligosaccharide containing 1 or multiple N-acetyllactosamine units andor 1 or multiple lacto-N-biose units or an intermediate intooligosaccharide, fucosylated and sialylated versions thereof.

DETAILED DESCRIPTION

According to a first aspect, the disclosure provides a method for theproduction of a di- or oligosaccharide having an N-acetylglucosamine(GlcNAc) unit at the reducing end by a cell, preferably a single cell.The method comprises the steps of:

-   -   providing a cell capable of synthesizing a nucleotide-sugar and        the monosaccharide GlcNAc and capable of glycosylating the        GlcNAc monosaccharide,    -   cultivating the cell under conditions permissive for producing        the di- or oligosaccharide,    -   preferably, separating the di- or oligosaccharide from the        cultivation.

In the scope of the disclosure, the wording “conditions permissive forproducing the di- or oligosaccharide” is to be understood to beconditions relating to physical or chemical parameters including but notlimited to temperature, pH, pressure, osmotic pressure andproduct/precursor/acceptor concentration.

In a particular embodiment, such conditions may include atemperature-range of 30+/−20 degrees centigrade, a pH-range of 7+/−3.

Throughout the disclosure, the feature “di- or oligosaccharide” ispreferably replaced with “oligosaccharide,” the feature “di- and/oroligosaccharides” is preferably replaced with “oligosaccharides.”According to the disclosure, the cell is capable to synthesize GlcNAcand this GlcNAc monosaccharide is further modified by glycosylationwhich is performed in the same cell resulting in the synthesis of a di-or oligosaccharide having GlcNAc at the reducing end. Hereby, the cellexpresses a glycosyltransferase to glycosylate the synthesizedN-acetylglucosamine to form a di- or oligosaccharide with GlcNAc at thereducing end from disclosure.

As such, the disclosure provides a method for the production of a di- oroligosaccharide having an N-acetylglucosamine (GlcNAc) unit at thereducing end by a cell, preferably a single cell. The method comprisesthe steps of:

-   -   providing a cell capable of synthesizing a nucleotide-sugar and        the monosaccharide GlcNAc and capable of expressing a        glycosyltransferase to glycosylate the GlcNAc monosaccharide to        form the di- or oligosaccharide,    -   cultivating the cell under conditions permissive for producing        the di- or oligosaccharide,    -   preferably, separating the di- or oligosaccharide from the        cultivation.

Glycosyltransferases are enzymes that catalyze the transfer of sugarmoieties from activated donor molecules to specific acceptor molecules,forming glycosidic bonds. As used herein the glycosyltransferase can beselected from the list comprising but not limited to:alpha-1,2-fucosyltransferases, alpha-1,3/1,4-fucosyltransferases,alpha-1,6-fucosyltransferases, alpha-2,3-sialyltransferases,alpha-2,6-sialyltransferases, alpha-2,8-sialyltransferases,beta-1,3-galactosyltransferases, beta-1,4-galactosyltransferases,alpha-1,3-galactosyltransferases, alpha-1,4-galactosyltransferases,N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases,glucosyltransferases, mannosyltransferases,N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyl transferases, glucosaminyltransferases,N-glycolylneuraminyltransferases.

The cell needs to produce GlcNAc intracellularly in order to be capableof further glycosylation of the synthesized GlcNAc. Intracellularproduction of GlcNAc needs to be understood as synthesis of GlcNAcinside the cell or inside the cell's cytoplasm, and not in an organelleor organelle membranes or the periplasm or cell membrane or cell wall ofthe cell.

In a preferred embodiment, the cell described herein expresses at leastone glucosamine 6-phosphate N-acetyltransferase and a phosphatase tosynthesize N-acetylglucosamine. In this preferred embodiment, theglucosamine 6-phosphate N-acetyltransferase is an enzyme capable ofconverting glucosamine-6-phosphate to N-acetylglucosamine-6-phosphate inthe cell and the phosphatase is capable to dephosphorylateN-acetylglucosamine-6-phosphate to produce N-acetylglucosamine in thecell. In a more preferred embodiment, this phosphatase is a HAD-likephosphatase. Phosphatases from the HAD superfamily and the HAD-likefamily are described in the art. Examples from these families can befound in the enzymes expressed from genes yqaB, inhX, yniC, ybiV, yidA,ybjI, yigL or cof from Escherichia coli or any one or more of e.g., theE. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP,YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV,YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU; or PsMupP from Pseudomonasputida, ScDOG1 from S. cerevisiae or BsAraL from Bacillus subtilis asdescribed in WO 2018122225. One phosphatase that catalyzes this reactionis identified in Blastocladiella emersonii. Phosphatases are generallynot specific and the activity is generally not related to the family orstructure. Other examples can thus be found in all phosphatase families.Specific phosphatases are easily identified and screened by well-knownmethods as described by Fahs et al. (ACS Chem. Biol. 11(11), 2944-2961(2016)).

In the context of the disclosure, it should be understood that the di-or oligosaccharide having a GlcNAc unit at the reducing end according tothe disclosure is synthesized intracellularly. The skilled person willfurther understand that a fraction or substantially all of thesynthesized di- or oligosaccharide having a GlcNAc unit at the reducingend remains intracellularly and/or is excreted outside the cell viapassive or active transport.

In a preferred embodiment, the cell is capable of expressing at leastone glycosyltransferase to glycosylate the GlcNAc monosaccharide to formthe di- or oligosaccharide. In another preferred embodiment, the cell iscapable of expressing at least two, more preferably at least three, evenmore preferably at least four, even more preferably at least five, mostpreferably at least 6, glycosyltransferases to glycosylate the GlcNAcmonosaccharide to form the di- or oligosaccharide according to thedisclosure.

In the context of the disclosure, the nucleotide-sugar is preferablydonor for the glycosyltransferase(s). Preferably, the cell is capable ofsynthesizing at least two, more preferably at least three, even morepreferably at least four, most preferably at least fivenucleotide-sugars.

In a preferred embodiment, the di- or oligosaccharide is lacto-N-biose(LNB) or N-acetyllactosamine (LacNAc), preferably an oligosaccharidecontaining LNB or LacNAc at the reducing end, more preferably sialylatedand/or fucosylated and/or galactosylated and/or GlcNAc-modified forms ofLNB or LacNAc, or even more preferably sialylated and/or fucosylatedand/or galactosylated and/or GlcNAc-modified forms of an oligosaccharidecontaining LNB or LacNAc at the reducing end. In another preferredembodiment, the di- or oligosaccharide is a neutral di- oroligosaccharide having an N-acetylglucosamine unit at the reducing end,preferably a neutral oligosaccharide containing LNB or LacNAc at thereducing end. Preferably, the neutral oligosaccharide is fucosylated.Alternatively, it is preferred that the neutral oligosaccharide is notfucosylated.

In another preferred embodiment, the disclosure provides a method forthe production of a mixture of di- and/or oligosaccharides having anN-acetylglucosamine (GlcNAc) unit at their reducing end by a cell,preferably a single cell. The method comprises the steps of:

-   -   providing a cell capable of synthesizing a nucleotide-sugar and        the monosaccharide GlcNAc and capable of expressing a        glycosyltransferase to glycosylate the GlcNAc monosaccharide to        form the mixture of di- and/or oligosaccharides having a GlcNAc        at their reducing end,    -   cultivating the cell under conditions permissive for producing        the mixture of di- and/or oligosaccharides having a GlcNAc at        their reducing end,    -   preferably, separating the mixture from the cultivation.

According to the disclosure, the mixture comprises or consists of atleast two different di- and/or oligosaccharides having a GlcNAc at theirreducing end, preferably at least three different di- and/oroligosaccharides having a GlcNAc at their reducing end, more preferablyat least four different di- and/or oligosaccharides having a GlcNAc attheir reducing end. Preferably, the mixture comprises or consists ofneutral di- and/or oligosaccharides. More preferably, the mixturecomprises or consists of charged and/or neutral di- and/oroligosaccharides. In a preferred embodiment of the method and/or cell,the charged di- and/or oligosaccharides are sialylated di- and/oroligosaccharides. In a preferred embodiment of the method and/or cell,the neutral di- and/or oligosaccharides are fucosylated. In anotherpreferred embodiment of the method and/or cell, the neutral di- and/oroligosaccharides are not fucosylated.

Also preferred within the scope of the disclosure, is a method for theproduction of a mixture comprising (i) a di- and/or oligosaccharidehaving an N-acetylglucosamine (GlcNAc) unit at the reducing end asdisclosed herein and (ii) one or more lactose-based mammalian milkoligosaccharides (MMOs), preferably one or more lactose-based human milkoligosaccharides (HMOs), by a cell, preferably a single cell. The methodcomprises the steps of:

-   -   providing a cell (i) capable of synthesizing a nucleotide-sugar        and the monosaccharide GlcNAc and capable of expressing a        glycosyltransferase to glycosylate the GlcNAc monosaccharide to        produce the di- and/or oligosaccharide, and (ii) wherein the        cell is further capable of expressing one or more        glycosyltransferase(s) to glycosylate lactose to produce the one        or more lactose-based MMOs and wherein the cell is capable of        synthesizing one or more nucleotide-sugar(s) which is/are        donor(s) for the glycosyltransferase(s), wherein the lactose is        either made by the cell (preferably intracellularly) or is added        before and/or during cultivation,    -   cultivating the cell under conditions permissive for producing        the mixture comprising i) a di- and/or oligosaccharide and ii)        one or more lactose-based MMOs,    -   preferably, separating the mixture from the cultivation.

The skilled person will understand that one or more glycosyltransferasesthat are involved in the production of the di- and/or oligosaccharidehaving GlcNAc at the reducing end can be the same as that/thoseglycosyltransferase(s) glycosylating lactose to form one or morelactose-based MMO(s). Alternatively, the glycosyltransferase(s) involvedin the production of the di- and/or oligosaccharide having GlcNAc at thereducing end is/are different from the glycosyltransferase(s) involvedin the production of one or more lactose-based MMOs.

The skilled person will also understand that the one or morenucleotide-sugar(s) that are involved in the production of the di-and/or oligosaccharide having GlcNAc at the reducing end can be the sameas that/those nucleotide-sugar(s) involved in the production of one ormore lactose-based MMOs. Alternatively, the one or morenucleotide-sugar(s) that are involved in the production of the di-and/or oligosaccharide having GlcNAc at the reducing end is/aredifferent from that/those nucleotide-sugar(s) involved in the productionof one or more lactose-based MMOs.

Each embodiment disclosed in the context of a method and/or cell for theproduction of a di- or oligosaccharide having a GlcNAc unit at thereducing end and for the production of a mixture of di- and/oroligosaccharides having a GlcNAc unit at the reducing end is alsodisclosed in the context of a method for the production of a mixturecomprising (i) a di- and/or oligosaccharide having a GlcNAc unit at thereducing end and (ii) one or more lactose-based MMOs, preferably one ormore lactose-based HMOs.

Further, each embodiment disclosed herein in the context of a methodand/or cell for the production of a di- or oligosaccharide having aGlcNAc unit at the reducing end or a mixture comprising a di- and/oroligosaccharide having a GlcNAc unit at the reducing end is alsodisclosed for the production of one or more lactose-based MMOs. Forexample, the amount and identity of glycosyltransferases andnucleotide-sugars as disclosed for the production of a di- oroligosaccharide having a GlcNAc unit at the reducing end, can also beapplied in the context of producing one or more lactose-based MMOs. Thesame applies for the other aspects of the disclosure such as the use ofthe cell for the production of a di- or oligosaccharide having a GlcNAcunit at the reducing end.

Another embodiment provides a method for the production of the di- oroligosaccharide with a GlcNAc unit at the reducing end by a geneticallymodified cell, preferably a single genetically modified cell, comprisingthe steps of:

-   -   providing a genetically modified cell capable of synthesizing a        nucleotide-sugar and the monosaccharide GlcNAc and capable of        glycosylating the GlcNAc monosaccharide,    -   cultivating the cell under conditions permissive for producing        the di- or oligosaccharide,    -   preferably separating the di- or oligosaccharide from the        cultivation.

Throughout the disclosure, unless explicitly stated otherwise, a“genetically modified cell” or “metabolically engineered cell”preferably means a cell which is genetically modified or metabolicallyengineered, respectively, for the production of a di- or oligosaccharidehaving an N-acetylglucosamine unit at the reducing end according to thedisclosure.

According to a second aspect, a metabolically engineered cell isprovided which is capable (i) to synthesize a nucleotide-sugar, (ii) tosynthesize N-acetylglucosamine, and (iii) of glycosylating theN-acetylglucosamine monosaccharide, wherein the cell produces a di- oroligosaccharide having an N-acetylglucosamine unit at the reducing end(or a mixture as disclosed herein), i.e., the cell is metabolicallyengineered for the production of the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end (or a mixture as disclosedherein). In the context of the disclosure, the di- or oligosaccharidehaving an N-acetylglucosamine unit at the reducing end (or mixture asdisclosed herein) preferably does not occur in the wild type progenitorof the metabolically engineered cell.

As such, the disclosure provides a cell which is metabolicallyengineered for the production of a di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end, wherein the cell iscapable (i) to synthesize a nucleotide-sugar, (ii) to synthesizeN-acetylglucosamine, and (iii) to express a glycosyltransferase toglycosylate the GlcNAc monosaccharide to form the di- oroligosaccharide. Each embodiment of the first aspect of the disclosurewhich further specifies any feature of the second aspect (such as theamount and identity of the nucleotide-sugar and glycosyltransferase;amount and identity of the di- or oligosaccharide, including themixtures thereof; etc.) is considered to be disclosed as well in thecontext of the second aspect.

In the method and cell described herein, the cell preferably comprisesmultiple copies of the same coding DNA sequence encoding for oneprotein. In the context of the disclosure, the protein can be aglycosyltransferase, a membrane transporter protein or any other proteinas disclosed herein. Throughout the disclosure, the feature “multiple”means at least 2, preferably at least 3, more preferably at least 4,even more preferably at least 5.

In the method and cell described herein, the cell is preferablygenetically modified in the expression or activity of an enzyme selectedfrom the group: a glucosamine 6-phosphate N-acetyltransferase, aphosphatase, a glycosyltransferase, anL-glutamine-D-fructose-6-phosphate aminotransferase, or an UDP-glucose4-epimerase. According to the disclosure, the as such enlisted enzymescomprising glucosamine 6-phosphate N-acetyltransferase, a phosphatase, aglycosyltransferase, an L-glutamine-D-fructose-6-phosphateaminotransferase, or an UDP-glucose 4-epimerase are either endogenousproteins with a modified expression or activity, preferably theendogenous proteins are overexpressed; or the enzymes of the as suchenlisted group are heterologous proteins, which can be heterologouslyexpressed by the cell. The heterologously expressed proteins will thenbe introduced and expressed, preferably overexpressed. In anotherembodiment, the endogenous proteins can have a modified expression inthe cell which also expresses a heterologous protein. Heterologousexpression can either be from the host's genome or from a vectorintroduced in the cell as described herein.

The as such enlisted enzymes comprising a glucosamine 6-phosphateN-acetyltransferase, a phosphatase, a glycosyltransferase, anL-glutamine-D-fructose-6-phosphate aminotransferase, or an UDP-glucose4-epimerase may be produced by expression by polynucleotides producedvia recombinant DNA technology using techniques well known in the art.Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences for thepolypeptides of disclosure and appropriate transcriptional and/ortranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. See, for example, the techniques described inSambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rdEdition, Cold Spring Harbor Laboratory Press, CSH, New York or toCurrent Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989and yearly updates).

According to another aspect of the disclosure, a vector can be providedto the cell, containing a polynucleotide encoding the as such enlistedenzymes comprising a glucosamine 6-phosphate N-acetyltransferase, aphosphatase, a glycosyltransferase, anL-glutamine-D-fructose-6-phosphate aminotransferase, or an UDP-glucose4-epimerase as described herein, wherein the polynucleotide is operablylinked to control sequences recognized by a cell transformed with thevector. In a particularly preferred embodiment, the vector is anexpression vector, and, according to another aspect of the disclosure,the vector can be present in the form of a plasmid, cosmid, phage,liposome, or virus. Thus, the polynucleotide encoding the polypeptidesof disclosure, may, e.g., be comprised in a vector which is to be stablytransformed/transfected into cells. In the vector, the coding sequenceof the polypeptides as described herein is under control of a promoter.The promoter can be e.g., an inducible promoter, so that the expressionof the gene/polynucleotide can be specifically targeted, and, ifdesired, the gene may be overexpressed in that way. The promoter canalso be a constitutive promoter. A great variety of expression systemscan be used to produce the polypeptides of the disclosure. Such vectorsinclude, among others, chromosomal, episomal and virus-derived vectors,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Thesevectors may contain selection markers such as but not limited toantibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNAsense/antisense markers. The expression system constructs may containcontrol regions that regulate as well as engender expression. Generally,any system or vector suitable to maintain, propagate or expresspolynucleotides and/or to express a polypeptide in a host may be usedfor expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., see above. For recombinant production, cells can begenetically engineered to incorporate expression systems or portionsthereof or polynucleotides of the disclosure. Introduction of apolynucleotide into the cell can be effected by methods described inmany standard laboratory manuals, such as Davis et al., Basic Methods inMolecular Biology, (1986), and Sambrook et al., 1989, supra.

According to a further aspect of the disclosure, the polynucleotidesencoding a glucosamine 6-phosphate N-acetyltransferase, a phosphatase, aglycosyltransferase, an L-glutamine-D-fructose-6-phosphateaminotransferase, or an UDP-glucose 4-epimerase are adapted to the codonusage of the respective cell or expression system.

In another embodiment, the cell used herein comprises anN-acetylglucosamine b-1,3-galactosyltransferase or anN-acetylglucosamine b-1,4-galactosyltransferase. The N-acetylglucosamineb-1,3-galactosyltransferase and the N-acetylglucosamineb-1,4-galactosyltransferase enzymes are glycosyltransferases capable oftransferring the galactose unit from an UDP-galactose donor to theGlcNAc acceptor in a beta-1,3- and beta-1,4-dependent glycosidiclinkage, respectively.

In another preferred embodiment, the cell used herein, whethergenetically modified or not, is capable to produce a nucleotide-sugarselected from the group: UDP-galactose (UDP-Gal),UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine(UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose(GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, or UDP-xylose. In a further embodiment, the cell isgenetically modified for the production of the nucleotide-sugar. Inanother preferred embodiment the cell is genetically modified for theoptimized production of the nucleotide-sugar.

In another embodiment, the cell is capable to produce UDP-galactose. Inan optional embodiment the cell is optimized for UDP-galactoseproduction. In an optional embodiment, the cell is modified in theexpression or activity of the UDP-glucose 4-epimerase GalE, which iscapable to convert UDP-glucose into UDP-galactose.

In a further embodiment, the nucleotide-sugar synthesized by the cell isUDP-galactose and the glycosyltransferase is an N-acetylglucosamineb-1,3-galactosyltransferase or an N-acetylglucosamineb-1,4-galactosyltransferase.

In a preferred embodiment, the disaccharide produced in the cell islacto-N-biose (Gal-b1,3-GlcNAc) or N-acetyllactosamine(Gal-b1,4-GlcNAc), which contain a galactose unit at the non-reducingend linked in a beta-1,3- or beta-1,4-dependent glycosidic linkage,respectively, to a GlcNAc moiety that is present at the reducing end ofthe disaccharide. In another preferred embodiment, the oligosaccharideproduced in the cell contains a lacto-N-biose (Gal-b1,3-GlcNAc) or anN-acetyllactosamine (Gal-b1,4-GlcNAc) at the reducing end.

In another preferred embodiment, the di- or oligosaccharide (or mixtureas described herein) synthesized by the cell according to the disclosuredoes not comprise a chitobiose (i.e., GlcNAc-b1,4-GlcNAc) at thereducing end, more preferably does not comprise a N-glycan. In otherwords, the cell is genetically modified for the production of a di- oroligosaccharide (or mixture as described herein) having anN-acetylglucosamine (GlcNAc) unit at the reducing end, wherein the di-or oligosaccharide does not comprise a chitobiose at the reducing end,more preferably does not comprise a N-glycan.

In another preferred embodiment, the N-acetylglucosamineb-1,3-galactosyltransferase expressed in the cell 1) has the PFAM domainPF00535 and (i) comprises the sequence [AGPS]XXLN(Xn)RXDXD with SEQ IDNO:01, wherein X is any amino acid and wherein n is 12 to 17, or (ii)comprises the sequence PXXLN(Xn)RXDXD(Xm)[FWY]XX[HKR]XX[NQST] with SEQID NO:02, wherein X is any amino acid and wherein n is 12 to 17 and m100 to 115, or (iii) comprises a polypeptide sequence according to anyone of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably any one of SEQID NO:03, 04, 05,06 or 07, more preferably any one of SEQ ID NO:03, 06or 07, most preferably any one of SEQ ID NO:03 or 06, or (iv) is afunctional homologue, variant or derivative of any one of SEQ ID NO: 03,04, 05, 06, 07 or 08, preferably of any one of SEQ ID NO:03, 04, 05,06or 07, more preferably of any one of SEQ ID NO:03, 06 or 07, mostpreferably of any one of SEQ ID NO:03 or 06, having at least 80% overallsequence identity to the full-length of any one of theN-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ IDNO: 03, 04, 05, 06, 07 or 08, preferably any one of SEQ ID NO:03, 04,05, 06 or 07, more preferably any one of SEQ ID NO:03, 06 or 07, mostpreferably any one of SEQ ID NO:03 or 06, and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or (v) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20consecutive amino acid residues from any one of SEQ ID NOs: 03, 04, 05,06, 07 or 08, preferably any one of SEQ ID NO:03, 04, 05, 06 or 07, morepreferably any one of SEQ ID NO:03, 06 or 07, most preferably any one ofSEQ ID NO:03 or 06, and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or (vi) is a functional fragmentof any one of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any one ofSEQ ID NO:03, 04, 05,06 or 07, more preferably any one of SEQ ID NO:03,06 or 07, most preferably any one of SEQ ID NO:03 or 06, and havingN-acetylglucosamine b-1,3-galactosyltransferase activity, or (vii)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NO: 03, 04, 05, 06, 07 or 08,preferably any one of SEQ ID NO:03, 04, 05, 06 or 07, more preferablyany one of SEQ ID NO:03, 06 or 07, most preferably any one of SEQ IDNO:03 or 06, and having N-acetylglucosamine b-1,3-galactosyltransferaseactivity, or 2) has the PFAM domain IPR002659 and (i) comprises thesequence KT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G, S)(Xp)X(no F, H, W,Y)[DE]D[ILV]XX[AG] with SEQ ID NO:09, wherein X is any amino acid andwherein n is 13 to 16, m 35 to 70 and p 20 to 45, or (ii) comprises apolypeptide sequence according to any one of SEQ ID NO: 10, 11, 12 or13, or (iii) is a functional homologue, variant or derivative of any oneof SEQ ID NO: 10, 11, 12 or 13 having at least 80% overall sequenceidentity to the full-length of any one of the N-acetylglucosamineb-1,3-galactosyltransferase polypeptide with SEQ ID NO: 10, 11, 12 or 13and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or(iv) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from anyone of SEQ ID NO: 10, 11, 12 or 13 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or (v) is a functional fragment ofany one of SEQ ID NO: 10, 11, 12 or 13 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or (vi) comprises a polypeptidecomprising or consisting of an amino acid sequence having at least 80%sequence identity to the full-length amino acid sequence of any one ofSEQ ID NO: 10, 11, 12 or 13 and having N-acetylglucosamineb-1,3-galactosyltransferase activity.

In another preferred embodiment, the N-acetylglucosamineb-1,4-galactosyltransferase expressed in the cell 1) has the PFAM domainPF01755 and (i) comprises the sequenceEXXCXXSHXX[ILV][FWY](Xn)EDD(Xm)[ACGST]XXYX[ILMV] with SEQ ID NO:14,wherein X is any amino acid and wherein n is 13 to 15 and m 50 to 76, or(ii) comprises a polypeptide sequence according to any one of SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably any one of SEQ IDNO: 15, 16, 17, 18, 20 or 21, more preferably any one of SEQ ID NO: 17,18, 20 or 21, or (iii) is a functional homologue, variant or derivativeof any one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23,preferably any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, morepreferably any one of SEQ ID NO: 17, 18, 20 or 21, having at least 80%overall sequence identity to the full-length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably any one of SEQ IDNO: 15, 16, 17, 18, 20 or 21, more preferably any one of SEQ ID NO: 17,18, 20 or 21, and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, (iv) or comprises an oligopeptide sequence of at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acidresidues from any one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or23, preferably any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, morepreferably any one of SEQ ID NO: 17, 18, 20 or 21, and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or (v) is afunctional fragment of any one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21,22 or 23, preferably any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21,more preferably any one of SEQ ID NO: 17, 18, 20 or 21, and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or23, preferably any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, morepreferably any one of SEQ ID NO: 17, 18, 20 or 21, and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or 2) has thePFAM domain PF00535 and (i) comprises the sequenceR[KN]XXXXXXXGXXXX[FL]XDXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO:24,wherein X is any amino acid and wherein n is 50 to 75 and m 10 to 30, or(ii) comprises the sequenceR[KN]XXXXXXXGXXXXFXDXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO:25, wherein X is any amino acid and wherein n is50 to 75, m is 10 to 30 and p is 20 to 25, or (iii) comprises apolypeptide sequence according to any one of SEQ ID NO: 26, 27 or 28, or(iv) is a functional homologue, variant or derivative of any one of SEQID NO: 58, 59 or 60 having at least 80% overall sequence identity to thefull-length of any one of the N-acetylglucosamineb-1,4-galactosyltransferase polypeptide with SEQ ID NO: 26, 27 or 28 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or (v)comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one ofSEQ ID NO: 26, 27 or 28 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (vi) is a functional fragmentof any one of SEQ ID NO: 26, 27 or 28 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (vii) comprises a polypeptidecomprising or consisting of an amino acid sequence having at least 80%sequence identity to the full-length amino acid sequence of any one ofSEQ ID NO: 26, 27 or 28 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or 3) has the PFAM domain PF02709and not having PFAM domain PF00535 and (i) comprises the sequence[FWY]XX[FY][FWY](X23)[FWY][GQ]X[DE]D with SEQ ID NO:29, wherein X is anyamino acid, or (ii) comprises the sequence[PV]W[GHNP](Xn)[FWY][GQ]X[DE]D with SEQ ID NO:30, wherein X is any aminoacid and wherein n is 21 to 24, or (iii) comprises a polypeptidesequence according to any one of SEQ ID NO: 31, 32, 33, 34, 35 or 36, or(iv) is a functional homologue, variant or derivative of any one of SEQID NO: 31, 32, 33, 34, 35 or 36 having at least 80% overall sequenceidentity to the full-length of any one of the N-acetylglucosamineb-1,4-galactosyltransferase polypeptide with SEQ ID NO: 31, 32, 33, 34,35 or 36 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, or (v) comprises an oligopeptide sequence of at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acidresidues from any one of SEQ ID NO: 31, 32, 33, 34, 35 or 36 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi) is afunctional fragment of any one of SEQ ID NO: 31, 32, 33, 34, 35 or 36and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or(vii) comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NO: 31, 32, 33, 34, 35 or 36 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or 4)has the PFAM domain PF03808 and (i) comprises the sequence[ST][FHY]XN(Xn)DG(X16)[HKR]X[ST]FDXX[ST]XA with SEQ ID NO:37, wherein Xis any amino acid and wherein n is 20 to 25, or (ii) comprises thesequence [ST][FHY]XN(Xn)DG(X16)[HKR]X[ ST]FDXX[ST]XA(Xm)[HR]XG[FWY](Xp)GXGXXXQ[DE] with SEQ ID NO:38, wherein X is anyamino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30,or (iii) comprises a polypeptide sequence according to any one of SEQ IDNO: 39, 40 or 41, or (iv) is a functional homologue, variant orderivative of any one of SEQ ID NO: 39, 40 or 41 having at least 80%overall sequence identity to the full-length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNO: 39, 40 or 41 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (v) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20consecutive amino acid residues from any one of SEQ ID NO: 39, 40 or 41and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or(vi) is a functional fragment of any one of SEQ ID NO: 39, 40 or 41 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or(vii) comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NO: 39, 40 or 41 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity.

The PFAM motifs used herein, PF00535, IPR002659, PF01755, PF02709,PF03808 are protein domains as annotated in the PFAM database versionPfam 33.1 as released on Jun. 11, 2020. PF00535 is found in theglycosyltransferase 2 (GT2) family that comprises enzymes that transfersugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose orCDP-abequose, to a range of acceptors including cellulose, dolicholphosphate and teichoic acids.

IPR002659 is found in the glycosyltransferase family 31 (GH31) thatcomprises enzymes with a number of known activities includingN-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase(2.4.1.149), beta-1,3-galactosyltransferase (2.4.1), fucose-specificbeta-1,3-N-acetylglucosaminyltransferase (2.4.1), globotriosylceramidebeta-1,3-GalNAc transferase (2.4.1.79). PF01755 is found in theglycosyltransferase 25 (GT25) family. This is a family ofglycosyltransferases involved in lipopolysaccharide (LPS) biosynthesis.These enzymes catalyze the transfer of various sugars onto the growingLPS chain during its biosynthesis. PF02709 refers to the Glyco_transf_7Cfamily. This is the N-terminal domain of a family ofgalactosyltransferases from a wide range of Metazoa with three relatedgalactosyltransferases activities, all three of which are possessed byone sequence in some cases: EC:2.4.1.90, N-acetyllactosamine synthase,EC:2.4.1.38, Beta-N-acetylglucosaminyl-glycopeptidebeta-1,4-galactosyltransferase, and EC:2.4.1.22 Lactose synthase.PF03808 refers to the glycosyltransferase 26 (GT26) family thatcomprises enzymes with activities like β-N-acetylmannosaminuronyltransferase (EC 2.4.1.-),β-N-acetyl-mannosaminyltransferase (EC 2.4.1.-),β-1,4-glucosyltransferase (EC 2.4.1.-) and β-1,4-galactosyltransferase(EC 2.4.1.-).

Proteins having the same PFAM domain and motifs as specified for eachclass of the N-acetylglucosamine b-1,3-galactosyltransferase or theN-acetylglucosamine b-1,4-galactosyltransferase can be searched via aRegEx analysis.

A RegEx, or Regular Expression, is a special sequence of characters thathelps to match or find other strings or sets of strings, using aspecialized syntax held in a pattern. Many programs are available to doRegEx search. One of them is the Python module “re” which provides fullsupport for regular expressions in Python. Detailed information, andknown by the persons skilled in the art, is available fromtowardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2,as released on 6 Apr. 2019. RegEx analyses for the proteins of thedisclosure have been included in the Examples part herein.

The glycosyltransferase family is a very broad family of enzymes capableto catalyze the transfer of sugar moieties from activated donormolecules to specific acceptor molecules, forming glycosidic bonds. Aclassification of glycosyltransferases using nucleotide diphospho-sugar,nucleotide monophospho-sugar and sugar phosphates and related proteinsinto distinct sequence-based families has been described (Campbell etal., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy(CArbohydrate-Active EnZymes) website (www.cazy.org).

In another preferred embodiment, the glucosamine 6-phosphateN-acetyltransferase is encoded by a heterologous nucleic acid. In otherwords, the glucosamine 6-phosphate N-acetyltransferase is heterologouslyexpressed in the cell. In another preferred embodiment, the glucosamine6-phosphate N-acetyltransferase (i) comprises, preferably is apolypeptide sequence with UniProt ID P43577 from Saccharomycescerevisiae, or (ii) is a functional homologue, variant or derivative ofthe polypeptide with UniProt ID P43577 having at least 80% overallsequence identity to the full-length of the polypeptide with UniProt IDP43577 and having glucosamine 6-phosphate N-acetyltransferase activity,or (iii) is a functional fragment of the polypeptide with UniProt IDP43577 and having glucosamine 6-phosphate N-acetyltransferase activity,or (iv) comprises a polypeptide comprising or consisting of an aminoacid sequence having at least 80% sequence identity to the full-lengthamino acid sequence of the polypeptide with UniProt ID P43577 and havingglucosamine 6-phosphate N-acetyltransferase activity. In anotherpreferred embodiment, the L-glutamine-D-fructose-6-phosphateaminotransferase (i) comprises, preferably is a polypeptide sequencewith UniProt ID P17169 from E. coli, or (ii) is a functional homologue,variant or derivative of the polypeptide with UniProt ID P17169 havingat least 80% overall sequence identity to the full-length of thepolypeptide with UniProt ID P17169 and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity, or (iii)is a functional fragment of the polypeptide with UniProt ID P17169 andhaving L-glutamine-D-fructose-6-phosphate aminotransferase activity, or(iv) comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of the polypeptide with UniProt ID P17169 and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity. In analternative preferred embodiment, the L-glutamine-D-fructose-6-phosphateaminotransferase (i) comprises, preferably is a polypeptide sequencediffering from the wild-type E. coli protein with UniProt ID P17169 byan A39T, an R250C and an G472S mutation as described by Deng et al.(Biochimie 88, 419-29 (2006), or (ii) is a functional homologue, variantor derivative of the mutant polypeptide (differing from the wild-type E.coli protein with UniProt ID P17169 by an A39T, an R250C and an G472Smutation) having at least 80% overall sequence identity to thefull-length of the mutant polypeptide (differing from the wild-type E.coli protein with UniProt ID P17169 by an A39T, an R250C and an G472Smutation) and having L-glutamine-D-fructose-6-phosphate aminotransferaseactivity, or (iii) is a functional fragment of the mutant polypeptide(differing from the wild-type E. coli protein with UniProt ID P17169 byan A39T, an R250C and an G472S mutation) and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity, or (iv)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of the mutant polypeptide (differing from the wild-type E.coli protein with UniProt ID P17169 by an A39T, an R250C and an G472Smutation) and having L-glutamine-D-fructose-6-phosphate aminotransferaseactivity.

The overall sequence identity is determined using a global alignmentalgorithm, such as the Needleman Wunsch algorithm in the program GAP(GCG Wisconsin Package, Accelrys), preferably with default parametersand preferably with sequences of mature proteins (i.e., without takinginto account secretion signals or transit peptides). Compared to overallsequence identity, the sequence identity will generally be higher whenonly conserved domains or motifs are considered.

At least 80% overall sequence identity to the full length of any one ofthe polypeptides with SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12, 13,15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36,39, 40 or 41 or UniProt IDs P43577 or P17169 or the mutant polypeptidediffering from UniProt ID P17169 by an A39T, an R250C and an G4725mutation should be understood as at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%overall sequence identity to any one of the polypeptides with SEQ ID NO:03, 04, 05, 06, 07, 08, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22,23, 26, 27, 28, 31, 32, 33, 34, 35, 36, 39, 40 or 41 or UniProt IDsP43577 or P17169 or the mutant polypeptide differing from UniProt IDP17169 by an A39T, an R250C and an G4725 mutation, respectively as givenherein. An oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one ofthe polypeptides with SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12, 13,15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36,39, 40 or 41 or UniProt IDs P43577 or P17169 or the mutant polypeptidediffering from UniProt ID P17169 by an A39T, an R250C and an G4725mutation should be understood as any one of oligopeptide sequences of atleast 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 up to the totalnumber of amino acid residues, of consecutive amino acid residues fromany one of the polypeptides with SEQ ID NO: 03, 04, 05, 06, 07, 08, 10,11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33,34, 35, 36, 39, 40 or 41 or UniProt IDs P43577 or P17169 or the mutantpolypeptide differing from UniProt ID P17169 by an A39T, an R250C and anG4725 mutation, respectively, preferably wherein the oligopeptide doesnot fully overlap with a PFAM domain if present, more preferably whereinthe oligopeptide does not overlap with a PFAM domain if present.

In a preferred embodiment of the method and/or cell of the disclosure,the cell is capable to catabolize a carbon source selected from the listcomprising: glucose, fructose, galactose, lactose, sucrose, maltose,malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose,mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose,hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup,glycerol, acetate, citrate, lactate, pyruvate.

In a preferred embodiment of the method and/or cell of the disclosure,the cell uses lactose in a glycosylation reaction to produce anoligosaccharide, preferably a lactose-based MMO as described herein.Lactose can be produced by the cell (for example, by the cell'smetabolism and/or upon metabolically engineering the cell for thispurpose as known to the skilled person), preferably intracellularly, orcan be added to the cell which can import the lactose through passive oractive transport. Lactose production by a cell can be obtained byexpression of an N-acetylglucosamine beta-1,4-galactosyltransferase andan UDP-glucose 4-epimerase. More preferably, the cell is modified forenhanced lactose production. The modification can be any one or morechosen from the group comprising over-expression of anN-acetylglucosamine beta-1,4-galactosyltransferase, over-expression ofan UDP-glucose 4-epimerase.

In a preferred embodiment of the method and/or cell of disclosure, acell using lactose as acceptor in a glycosylation reaction preferablyhas a transporter for the uptake of lactose from the cultivation. Morepreferably, the cell is optimized for lactose uptake. The optimizationcan be over-expression of a lactose transporter like a lactose permeasefrom E. coli or Kluyveromyces lactis. It is preferred the cellconstitutively expresses the lactose permease. Lactose can be added atthe start of the cultivation or it can be added as soon as enoughbiomass has been formed during the growth phase of the cultivation,i.e., the lactose-based MIO production phase which is initiated by theaddition of lactose to the cultivation is decoupled from the growthphase. In a preferred embodiment, lactose is added at the start and/orduring the cultivation, i.e., the growth phase and production phase arenot decoupled.

In a preferred embodiment of the method and/or cell according to thedisclosure, the cell resists the phenomenon of lactose killing whengrown in an environment in which lactose is combined with one or moreother carbon source(s). With the term “lactose killing” is meant thehampered growth of the cell in medium in which lactose is presenttogether with another carbon source. In a preferred embodiment, the cellis genetically modified such that it retains at least 50% of the lactoseinflux without undergoing lactose killing, even at high lactoseconcentrations, as is described in WO 2016/075243. The geneticmodification comprises expression and/or over-expression of an exogenousand/or an endogenous lactose transporter gene by a heterologous promoterthat does not lead to a lactose killing phenotype and/or modification ofthe codon usage of the lactose transporter to create an alteredexpression of the lactose transporter that does not lead to a lactosekilling phenotype. The content of WO 2016/075243 in this regard isincorporated by reference.

In a further embodiment of the disclosure, fructose-6-phosphate issubstrate of the L-glutamine-D-fructose-6-phosphate aminotransferasewhich is expressed in the cell and which is capable of convertingfructose-6-phosphate into glucosamine-6-phosphate as precursor in thesynthesis toward GlcNAc. Glucosamine-6-phosphate is substrate of theglucosamine 6-phosphate N-acetyltransferase which is expressed in thecell and which is capable of converting glucosamine-6-phosphate toN-acetylglucosamine-6-phosphate. N-acetylglucosamine-6-phosphate issubstrate of a phosphatase which is expressed in the cell and which iscapable of dephosphorylating N-acetylglucosamine-6-phosphate tosynthesize the monosaccharide GlcNAc.

In another preferred embodiment, the cell is unable to convertN-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/orunable to convert glucosamine-6-phosphate to fructose-6-phoshate. In acell N-acetylglucosamine-6-phosphate can be converted toglucosamine-6-phosphate by activity of anN-acetylglucosamine-6-phosphate deacetylase like nagA andglucosamine-6-phosphate can be converted to fructose-6-phoshate byactivity of a glucosamine-6-phosphate deaminase like nagB. SuchN-acetylglucosamine-6-phosphate deacetylase and/orglucosamine-6-phosphate deaminase can obtain reduced expression orreduced activity or can be inactivated by mutagenesis or by partial orfull deletion of the corresponding polynucleotides encoding for thecoding sequences or by mutagenesis of the promoter sequences controllingthe expression of the corresponding encoding polynucleotides by methodswell-known in the art.

In one embodiment, the cell is capable to synthesize thenucleotide-sugar GDP-fucose. The GDP-fucose can be provided by an enzymeexpressed in the cell or by the metabolism of the cell. Such cellsynthesizing GDP-fucose can express an enzyme converting, e.g., fucose,which is to be added to the cell, to GDP-fucose. This enzyme may be,e.g., a bifunctional fucose kinase/fucose-1-phosphateguanylyltransferase, like Fkp from Bacteroides fragilis, or thecombination of one separate fucose kinase together with one separatefucose-1-phosphate guanylyltransferase like they are known from severalspecies including Homo sapiens, Sus scrofa and Rattus norvegicus.

Preferably, the cell is modified to produce GDP-fucose.

More preferably, the cell is modified for enhanced GDP-fucoseproduction. The modification can be any one or more chosen from thegroup comprising knock-out of an UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase encoding gene, over-expression of aGDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose4,6-dehydratase encoding gene, over-expression of a mannose-1-phosphateguanylyltransferase encoding gene, over-expression of aphosphomannomutase encoding gene and over-expression of amannose-6-phosphate isomerase encoding gene.

In one embodiment, the cell is capable to synthesize thenucleotide-sugar UDP-galactose. The UDP-galactose can be provided by anenzyme expressed in the cell or by the metabolism of the cell.Preferably, the cell is modified to synthesize UDP-galactose. Morepreferably, the cell is modified for enhanced UDP-galactose production.The modification can be one or more chosen from the group comprisingknock-out of an 5′-nucleotidase/UDP-sugar hydrolase encoding gene,knock-out of a galactose-1-phosphate uridylyltransferase encoding gene,and overexpression of a UDP-galactose 4-epimerase like galE.

In one embodiment, the cell is capable to synthesize thenucleotide-sugar CMP-N-acetylneuraminic acid (CMP-sialic acid). TheCMP-N-acetylneuraminic acid can be provided by an enzyme expressed inthe cell or by the metabolism of the cell. Preferably, the cell ismodified to synthesize CMP-N-acetylneuraminic acid. More preferably, thecell is modified for enhanced CMP-N-acetylneuraminic acid production.The modification can be one or more chosen from the group comprisingover-expression of a CMP-sialic acid synthetase encoding gene,over-expression of a sialate synthase encoding gene, and over-expressionof an N-acetyl-D-glucosamine 2-epimerase encoding gene.

Synthesis of CMP-N-acetylneuraminic acid makes use of GlcNAc but GlcNAcin the cell as described herein is used in the synthesis of a di- oroligosaccharide with a GlcNAc unit at the reducing end. Production ofCMP-N-acetylneuraminic acid in the cell may thus lower the GlcNAcavailable for the production of the bioproducts on interest, i.e., a di-or oligosaccharide with a GlcNAc unit at the reducing end. Production ofCMP-N-acetylneuraminic acid and of GlcNAc needs to be carefullyoptimized to each other to ensure high levels of bothCMP-N-acetylneuraminic acid and GlcNAc. Optimization may includeefficient balancing and fine-tuning of the expression levels ofpolypeptides involved in the synthesis of both CMP-N-acetylneuraminicacid and GlcNAc.

In another preferred embodiment, the cell expresses at least one furtherglycosyltransferase to glycosylate the synthesized N-acetylglucosaminemonosaccharide to form an oligosaccharide with GlcNAc at the reducingend as disclosed herein. Preferably, the cell expresses at least oneglycosyltransferase to glycosylate the GlcNAc monosaccharide to form thedi- or oligosaccharide. More preferably, the cell expresses at leasttwo, even more preferably at least three, even more preferably at leastfour glycosyltransferases, most preferably at least fiveglycosyltransferases to glycosylate the GlcNAc monosaccharide to formthe di- or oligosaccharide according to the disclosure. Theglycosyltransferase can be selected from the list comprising but notlimited to: alpha-1,2-fucosyltransferases,alpha-1,3/1,4-fucosyltransferases, alpha-1,6-fucosyltransferases,alpha-2,3-sialyltransferases, alpha-2,6-sialyltransferases,alpha-2,8-sialyltransferases, beta-1,3-galactosyltransferases,beta-1,4-galactosyltransferases, alpha-1,3-galactosyltransferases,alpha-1,4-galactosyltransferases, N-acetylglucosaminyltransferases,N-acetylgalactosaminyltransferases, glucosyltransferases,mannosyltransferases, N-acetylmannosaminyltransferases,xylosyltransferases, glucuronyl transferases, galacturonyl transferases,glucosaminyltransferases, N-glycolylneuraminyltransferases. In apreferred embodiment, the glycosyltransferase is an endogenous proteinof the cell with a modified expression or activity, preferably theendogenous glycosyltransferase is overexpressed; alternatively theglycosyltransferase is a heterologous protein that is heterogeneouslyintroduced and expressed in the cell, preferably overexpressed. Theendogenous glycosyltransferase can have a modified expression in thecell which also expresses a heterologous glycosyltransferase. The assuch synthesized oligosaccharides can be of the linear type or of thebranched type and can contain multiple monosaccharide building blocks asexplained in the definitions as described herein.

By combining different active glycosyltransferases in the same cell asdescribed herein, producing GlcNAc and nucleotide-activated sugarscomprising UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine(UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc),UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose(GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, or UDP-xylose, the cell is capable to produce a di- oroligosaccharide with a GlcNAc unit at the reducing end according to theformula 1:

The oligosaccharide as described herein can be formed in a linear or ina branched structure, containing both alpha- and beta-glycosidic bondsor can contain only beta-glycosidic bonds, wherein B isN-acetylglucosamine, and wherein A, V, W, X, Y, and/or Z are absent, orare a galactose, glucose, fucose, mannose, xylose, glucuronic acid,galacturonic acid, iduronic acid, N-acetylneuraminic acid,N-glycolylneuraminic acid, glucosamine, N-acetylgalactosamine,N-acetylmannosamine, N-acetylglucosamine and/or an oligosaccharidestructure as defined by the formula 2:

wherein B is N-acetylglucosamine, and wherein A, V, W, X, Y, and/or Zare absent, or are a galactose, glucose, fucose, mannose, xylose,glucuronic acid, galacturonic acid, iduronic acid, N-acetylneuraminicacid, N-glycolylneuraminic acid, glucosamine, N-acetylgalactosamine,N-acetylmannosamine or N-acetylglucosamine, and wherein, in formula 1and in formula 2, m is 3 with optionally v is 4, or m is 4 withoptionally v is 3, and wherein p is 3 and/or 4, and wherein w is 6, andwherein x is 3 and X is no monosaccharide if n>1 and p is 3, and whereiny is 4 and Y is no monosaccharide if n>1 and p is 4, and wherein z is 6,and wherein n ranges from 1 to 10.

In a more preferred embodiment, the cell as described herein is modifiedin the expression or activity of the further glycosyltransferase.

In another preferred embodiment, the oligosaccharide with a GlcNAc unitat the reducing end produced in the method as described herein and bythe cell as described herein is chosen from the list comprising:2-fucosyl lacto-N-biose, 4-fucosyl lacto-N-biose, 2-4-difucosyllacto-N-biose, 3′-sialyl lacto-N-biose, 6′-sialyl lacto-N-biose,3′,6′-disialyl lacto-N-biose, 6,6′-disialyl lacto-N-biose,2′-fucosyl-3′-sialyl lacto-N-biose, 2′-fucosyl-6′-sialyl lacto-N-biose,4-fucosyl-3′-sialyl lacto-N-biose, 4-fucosyl-6′-sialyl lacto-N-biose,2-fucosyl N-acetyllactosamine, 3′-fucosyl N-acetyllactosamine,2,3′-difucosyl N-acetyllactosamine, 3′-sialyl N-acetyllactosamine,6′-sialyl N-acetyllactosamine, 3′,6′-disialyl N-acetyllactosamine,6,6′-disialyl N-acetyllactosamine, 2′-fucosyl-3′-sialylN-acetyllactosamine, 2′-fucosyl-6′-sialyl N-acetyllactosamine,3-fucosyl-3′-sialyl N-acetyllactosamine, 3′-fucosyl-6′-sialylN-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), thexenotransplantation epitope (Gal-a1,3-Gal-b1,4-GlcNAc),Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4-GlcNAc.

In a preferred embodiment of the method and/or cell according to thedisclosure, the cell expresses a membrane transporter protein or apolypeptide having transport activity hereby transporting compoundsacross the outer membrane of the cell wall. In a more preferredembodiment of the method and/or cell of the disclosure, the cell ismodified in the expression or activity of the membrane transporterprotein or polypeptide having transport activity. The membranetransporter protein or polypeptide having transport activity is anendogenous protein of the cell with a modified expression or activity,preferably the endogenous membrane transporter protein or polypeptidehaving transport activity is overexpressed; alternatively the membranetransporter protein or polypeptide having transport activity is aheterologous protein that is heterogeneously introduced and expressed inthe cell, preferably overexpressed. The endogenous membrane transporterprotein or polypeptide having transport activity can have a modifiedexpression in the cell which also expresses a heterologous membranetransporter protein or polypeptide having transport activity.

In a more preferred embodiment of the method and/or cell of thedisclosure, the membrane transporter protein or polypeptide havingtransport activity is chosen from the list comprising porters,P—P-bond-hydrolysis-driven transporters, β-barrel porins, auxiliarytransport proteins, putative transport proteins andphosphotransfer-driven group translocators. In an even more preferredembodiment of the method and/or cell of the disclosure, the porterscomprise MFS transporters, sugar efflux transporters and siderophoreexporters. In another more preferred embodiment of the method and/orcell of the disclosure, the P—P-bond-hydrolysis-driven transporterscomprise ABC transporters and siderophore exporters.

In another preferred embodiment of the method and/or cell of thedisclosure, the membrane transporter protein or polypeptide havingtransport activity controls the flow over the outer membrane of the cellwall of the di- or oligosaccharide having a GlcNAc unit at the reducingend. In an alternative and or additional preferred embodiment of themethod and/or cell of the disclosure, the membrane transporter proteinor polypeptide having transport activity controls the flow over theouter membrane of the cell wall of the di- and oligosaccharide having aGlcNAc unit at the reducing end. In an alternative and or additionalpreferred embodiment of the method and/or cell of the disclosure, themembrane transporter protein or polypeptide having transport activitycontrols the flow over the outer membrane of the cell wall of the one ormore lactose-based MMO(s).

In an alternative and/or additional preferred embodiment of the methodand/or cell of the disclosure, the membrane transporter protein orpolypeptide having transport activity controls the flow over the outermembrane of the cell wall of one or more precursor(s) to be used in theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end In an alternative and/or additional preferred embodiment ofthe method and/or cell of the disclosure, the membrane transporterprotein or polypeptide having transport activity controls the flow overthe outer membrane of the cell wall of one or more precursor(s) to beused in the production of the di- and oligosaccharide having a GlcNAcunit at the reducing end. In an alternative and/or additional preferredembodiment of the method and/or cell of the disclosure, the membranetransporter protein or polypeptide having transport activity controlsthe flow over the outer membrane of the cell wall of one or moreprecursor(s) to be used in the production of the one or morelactose-based MMO(s).

In an alternative and/or additional preferred embodiment of the methodand/or cell of the disclosure, the membrane transporter protein orpolypeptide having transport activity controls the flow over the outermembrane of the cell wall of one or more acceptor(s) to be used in theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end. In an alternative and/or additional preferred embodimentof the method and/or cell of the disclosure, the membrane transporterprotein or polypeptide having transport activity controls the flow overthe outer membrane of the cell wall of one or more acceptor(s) to beused in the production of the di- and oligosaccharide having a GlcNAcunit at the reducing end. In an alternative and/or additional preferredembodiment of the method and/or cell of the disclosure, the membranetransporter protein or polypeptide having transport activity controlsthe flow over the outer membrane of the cell wall of one or moreacceptor(s) to be used in the production of the one or morelactose-based MMO(s).

In another preferred embodiment of the method and/or cell of thedisclosure, the membrane transporter protein or polypeptide havingtransport activity provides improved production of the di- oroligosaccharide having a GlcNAc unit at the reducing end. In anotherpreferred embodiment of the method and/or cell of the disclosure, themembrane transporter protein or polypeptide having transport activityprovides improved production of the di- and oligosaccharide having aGlcNAc unit at the reducing end. In another preferred embodiment of themethod and/or cell of the disclosure, the membrane transporter proteinor polypeptide having transport activity provides improved production ofthe one or more lactose-based MMO(s).

In an alternative and/or additional preferred embodiment of the methodand/or cell of the disclosure, the membrane transporter protein orpolypeptide having transport activity provides enabled efflux of the di-or oligosaccharide having GlcNAc residue at the reducing end. In analternative and/or additional preferred embodiment of the method and/orcell of the disclosure, the membrane transporter protein or polypeptidehaving transport activity provides enabled efflux of the di- andoligosaccharide having GlcNAc residue at the reducing end. In analternative and/or additional preferred embodiment of the method and/orcell of the disclosure, the membrane transporter protein or polypeptidehaving transport activity provides enabled efflux of the one or morelactose-based MMO(s).

In an alternative and/or additional preferred embodiment of the methodand/or cell of the disclosure, the membrane transporter protein orpolypeptide having transport activity provides enhanced efflux of thedi- or oligosaccharide having GlcNAc residue at the reducing end. In analternative and/or additional preferred embodiment of the method and/orcell of the disclosure, the membrane transporter protein or polypeptidehaving transport activity provides enhanced efflux of the di- andoligosaccharide having GlcNAc residue at the reducing end. In analternative and/or additional preferred embodiment of the method and/orcell of the disclosure, the membrane transporter protein or polypeptidehaving transport activity provides enhanced efflux of the one or morelactose-based MMO(s).

In another preferred embodiment of the method and/or cell of thedisclosure, the cell expresses a polypeptide selected from the groupcomprising a lactose transporter like e.g., the LacY or lac12 permease,a glucose transporter, a galactose transporter, a fucose transporter, atransporter for a nucleotide-activated sugar, for example, a transporterfor UDP-Gal, UDP-GlcNAc, GDP-Fuc or CMP-sialic acid. As such, thetransporter internalizes a to the medium added precursor and/or acceptorfor the synthesis of a di- or oligosaccharide having a GlcNAc unit atthe reducing end and/or a lactose-based MMO.

In a more preferred embodiment of the method and/or cell of thedisclosure, the cell expresses a membrane transporter protein belongingto the family of MFS transporters like e.g., an MdfA polypeptide of themultidrug transporter MdfA family from species comprising E. coli(UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9),Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei(UniProt ID G9Z5F4). In another more preferred embodiment of the methodand/or cell of the disclosure, the cell expresses a membrane transporterprotein belonging to the family of sugar efflux transporters like e.g.,a SetA polypeptide of the SetA family from species comprising E. coli(UniProt ID P31675), Citrobacter koseri (UniProt ID A0A078LM16) andKlebsiella pneumoniae (UniProt ID A0A0C4MGS7). In another more preferredembodiment of the method and/or cell of the disclosure, the cellexpresses a membrane transporter protein belonging to the family ofsiderophore exporters like e.g., the E. coli entS (UniProt ID P24077)and the E. coli iceT (UniProt ID A0A024L207). In another more preferredembodiment of the method and/or cell of the disclosure, the cellexpresses a membrane transporter protein belonging to the family of ABCtransporters like e.g., oppF from E. coli (UniProt ID P77737), lmrA fromLactococcus lactis subsp. lactis by. diacetylactis (UniProt IDA0A1V0NEL4) and Blon 2475 from Bifidobacterium longum subsp. infantis(UniProt ID B7GPD4).

According to another embodiment of the method and/or cell of thedisclosure, the cell is capable to produce phosphoenolpyruvate (PEP).According to another embodiment of the method and/or cell of thedisclosure, the cell comprises a pathway for production of a di- oroligosaccharide having an N-acetylglucosamine unit at the reducing endcomprising a pathway for production of PEP. In a preferred embodiment ofthe method and/or cell of the disclosure, the cell is modified forenhanced production and/or supply of PEP compared to a non-modifiedprogenitor.

In another preferred embodiment, the cell comprises a pathway forproduction of a di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end comprising any one or more modifications forenhanced production and/or supply of PEP compared to a non-modifiedprogenitor.

In a preferred embodiment and as a means for enhanced production and/orsupply of PEP, one or more PEP-dependent, sugar-transportingphosphotransferase system(s) is/are disrupted such as but not limitedto: 1) the N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193),which is, for instance, encoded by the nagE gene (or the clusternagABCD) in E. coli or Bacillus species, 2) ManXYZ which encodes theEnzyme 11 Man complex (mannose PTS permease,protein-Npi-phosphohistidine-D-mannose phosphotransferase) that importsexogenous hexoses (mannose, glucose, glucosamine, fructose,2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases thephosphate esters into the cell cytoplasm, 3) the glucose-specific PTStransporter (for instance, encoded by PtsG/Crr) which takes up glucoseand forms glucose-6-phosphate in the cytoplasm, 4) the sucrose-specificPTS transporter which takes up sucrose and forms sucrose-6-phosphate inthe cytoplasm, 5) the fructose-specific PTS transporter (for instance,encoded by the genes fruA and fruB and the kinase fruK which takes upfructose and forms in a first step fructose-1-phosphate and in a secondstep fructose1,6 bisphosphate, 6) the lactose PTS transporter (forinstance, encoded by lacE in Lactococcus casei) which takes up lactoseand forms lactose-6-phosphate, 7) the galactitol-specific PTS enzymewhich takes up galactitol and/or sorbitol and formsgalactitol-1-phosphate or sorbitol-6-phosphate respectively, 8) themannitol-specific PTS enzyme which takes up mannitol and/or sorbitol andforms mannitol-1-phosphate or sorbitol-6-phosphate respectively, and 9)the trehalose-specific PTS enzyme which takes up trehalose and formstrehalose-6-phosphate.

In another and/or additional preferred embodiment and as a means forenhanced production and/or supply of PEP, the full PTS system isdisrupted by disrupting the PtsIH/Crr gene cluster. PtsI (Enzyme I) is acytoplasmic protein that serves as the gateway for thephosphoenolpyruvate:sugar phosphotransferase system (PTSsugar) of E.coli K-12. PtsI is one of two (PtsI and PtsH) sugar non-specific proteinconstituents of the PTSsugar which along with a sugar-specific innermembrane permease effects a phosphotransfer cascade that results in thecoupled phosphorylation and transport of a variety of carbohydratesubstrates. HPr (histidine containing protein) is one of twosugar-non-specific protein constituents of the PTSsugar. It accepts aphosphoryl group from phosphorylated Enzyme I (PtsI-P) and thentransfers it to the EIIA domain of any one of the many sugar-specificenzymes (collectively known as Enzymes II) of the PTSsugar. Crr orEIIAGlc is phosphorylated by PEP in a reaction requiring PtsH and PtsI.

In another and/or additional preferred embodiment, the cell is furthermodified to compensate for the deletion of a PTS system of a carbonsource by the introduction and/or overexpression of the correspondingpermease. These are e.g., permeases or ABC transporters that comprisebut are not limited to transporters that specifically import lactosesuch as e.g., the transporter encoded by the LacY gene from E. coli,sucrose such as e.g., the transporter encoded by the cscB gene from E.coli, glucose such as e.g., the transporter encoded by the galP genefrom E. coli, fructose such as e.g., the transporter encoded by the fruIgene from Streptococcus mutans, or the Sorbitol/mannitol ABC transportersuch as the transporter encoded by the cluster SmoEFGK of Rhodobactersphaeroides, the trehalose/sucrose/maltose transporter such as thetransporter encoded by the gene cluster ThuEFGK of Sinorhizobiummeliloti and the N-acetylglucosamine/galactose/glucose transporter suchas the transporter encoded by NagP of Shewanella oneidensis. Examples ofcombinations of PTS deletions with overexpression of alternativetransporters are: 1) the deletion of the glucose PTS system, e.g., ptsGgene, combined with the introduction and/or overexpression of a glucosepermease (e.g., galP of glcP), 2) the deletion of the fructose PTSsystem, e.g., one or more of the fruB, fruA, fruK genes, combined withthe introduction and/or overexpression of fructose permease, e.g., fruI,3) the deletion of the lactose PTS system, combined with theintroduction and/or overexpression of lactose permease, e.g., LacY,and/or 4) the deletion of the sucrose PTS system, combined with theintroduction and/or overexpression of a sucrose permease, e.g., cscB.

In a further preferred embodiment, the cell is modified to compensatefor the deletion of a PTS system of a carbon source by the introductionof carbohydrate kinases, such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC2.7.1.63), galactokinase (EC 2.7.1.6), and/or fructokinase (EC 2.7.1.3,EC 2.7.1.4). Examples of combinations of PTS deletions withoverexpression of alternative transporters and a kinase are: 1) thedeletion of the glucose PTS system, e.g., ptsG gene, combined with theintroduction and/or overexpression of a glucose permease (e.g., galP ofglcP), combined with the introduction and/or overexpression of aglucokinase (e.g., glk), and/or 2) the deletion of the fructose PTSsystem, e.g., one or more of the fruB, fruA, fruK genes, combined withthe introduction and/or overexpression of fructose permease, e.g., fruI,combined with the introduction and/or overexpression of a fructokinase(e.g., frk or mak).

In another and/or additional preferred embodiment and as a means forenhanced production and/or supply of PEP, the cell is modified by theintroduction of or modification in any one or more of the listcomprising phosphoenolpyruvate synthase activity (EC: 2.7.9.2 encoded,for instance, in E. coli by ppsA), phosphoenolpyruvate carboxykinaseactivity (EC 4.1.1.32 or EC 4.1.1.49 encoded, for instance, inCorynebacterium glutamicum by PCK or in E. coli by pckA, resp.),phosphoenolpyruvate carboxylase activity (EC 4.1.1.31 encoded, forinstance, in E. coli by ppc), oxaloacetate decarboxylase activity (EC4.1.1.112 encoded, for instance, in E. coli by eda), pyruvate kinaseactivity (EC 2.7.1.40 encoded, for instance, in E. coli by pykA andpykF), pyruvate carboxylase activity (EC 6.4.1.1 encoded, for instance,in B. subtilis by pyc) and malate dehydrogenase activity (EC 1.1.1.38 orEC 1.1.1.40 encoded, for instance, in E. coli by maeA or maeB,respectively).

In a more preferred embodiment, the cell is modified to overexpress anyone or more of the polypeptides comprising ppsA from E. coli (UniProt IDP23538), PCK from C. glutamicum (UniProt ID Q6F5A5), pcka from E. coli(UniProt ID P22259), eda from E. coli (UniProt ID P0A955), maeA from E.coli (UniProt ID P26616) and maeB from E. coli (UniProt ID P76558).

In another and/or additional preferred embodiment, the cell is modifiedto express any one or more polypeptide having phosphoenolpyruvatesynthase activity, phosphoenolpyruvate carboxykinase activity,oxaloacetate decarboxylase activity, or malate dehydrogenase activity.

In another and/or additional preferred embodiment and as a means forenhanced production and/or supply of PEP, the cell is modified by areduced activity of phosphoenolpyruvate carboxylase activity, and/orpyruvate kinase activity, preferably a deletion of the genes encodingfor phosphoenolpyruvate carboxylase, the pyruvate carboxylase activityand/or pyruvate kinase.

In an exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate synthase combined with thedeletion of a phosphoenolpyruvate carboxylase gene, the overexpressionof phosphoenolpyruvate synthase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate carboxykinase combined with thedeletion of a phosphoenolpyruvate carboxylase gene, the overexpressionof phosphoenolpyruvate carboxykinase combined with the deletion of apyruvate carboxylase gene, the overexpression of oxaloacetatedecarboxylase combined with the deletion of a pyruvate kinase gene, theoverexpression of oxaloacetate decarboxylase combined with the deletionof a phosphoenolpyruvate carboxylase gene, the overexpression ofoxaloacetate decarboxylase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of malate dehydrogenase combinedwith the deletion of a pyruvate kinase gene, the overexpression ofmalate dehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene and/or the overexpression of malate dehydrogenasecombined with the deletion of a pyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase, the overexpression of phosphoenolpyruvate synthasecombined with the overexpression of an oxaloacetate decarboxylase, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a malate dehydrogenase, the overexpression ofphosphoenolpyruvate carboxykinase combined with the overexpression of anoxaloacetate decarboxylase, the overexpression of phosphoenolpyruvatecarboxykinase combined with the overexpression of a malatedehydrogenase, the overexpression of oxaloacetate decarboxylase combinedwith the overexpression of a malate dehydrogenase, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of anoxaloacetate decarboxylase, the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase and the overexpression of a malate dehydrogenase, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase, the overexpression of phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase and the overexpression of a malate dehydrogenase and/orthe overexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvate kinasegene, the overexpression of phosphoenolpyruvate carboxykinase combinedwith the overexpression of an oxaloacetate decarboxylase combined withthe deletion of a pyruvate kinase gene, the overexpression ofphosphoenolpyruvate carboxykinase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvate kinasegene, the overexpression of an oxaloacetate decarboxylase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene, the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase and the overexpression of an oxaloacetate decarboxylasecombined with the deletion of a pyruvate kinase gene, the overexpressionof phosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene, the overexpression of a phosphoenolpyruvate carboxykinasecombined with the overexpression of an oxaloacetate decarboxylase andthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene, the overexpression of phosphoenolpyruvatesynthase combined the overexpression of oxaloacetate decarboxylase andthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of phosphoenolpyruvate synthasecombined with the overexpression of an oxaloacetate decarboxylasecombined with the deletion of a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a malate dehydrogenase combined with the deletion of aphosphoenolpyruvate carboxylase gene, the overexpression of aphosphoenolpyruvate carboxykinase combined with the overexpression of anoxaloacetate decarboxylase combined with the deletion of aphosphoenolpyruvate carboxylase gene, the overexpression of aphosphoenolpyruvate carboxykinase combined with the overexpression of amalate dehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of an oxaloacetate decarboxylasecombined with the overexpression of a malate dehydrogenase combined withthe deletion of a phosphoenolpyruvate carboxylase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a phosphoenolpyruvate carboxylase gene, the overexpressionof phosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of phosphoenolpyruvate synthasecombined with the overexpression of a phosphoenolpyruvate carboxykinaseand the overexpression of an oxaloacetate decarboxylase and theoverexpression of a malate dehydrogenase combined with the deletion of aphosphoenolpyruvate carboxylase gene, the overexpression of aphosphoenolpyruvate carboxykinase combined with the overexpression of anoxaloacetate decarboxylase and the overexpression of a malatedehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of phosphoenolpyruvate synthasecombined the overexpression of an oxaloacetate decarboxylase and theoverexpression of a malate dehydrogenase combined with the deletion of aphosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase combined with the deletion of a pyruvate carboxylase gene,the overexpression of a phosphoenolpyruvate carboxykinase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate carboxylase gene, the overexpression of an oxaloacetatedecarboxylase combined with the overexpression of a malate dehydrogenasecombined with the deletion of a pyruvate carboxylase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase and the overexpression of a malate dehydrogenase combinedwith the deletion of a pyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of anoxaloacetate decarboxylase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene and aphosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of anoxaloacetate decarboxylase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a malate dehydrogenase combined with the deletion of apyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate carboxykinase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylasegene, the overexpression of a phosphoenolpyruvate carboxykinase combinedwith the overexpression of a malate dehydrogenase combined with thedeletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylasegene, the overexpression of an oxaloacetate decarboxylase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of a phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylasegene, the overexpression of a phosphoenolpyruvate synthase combined withthe overexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of a malate dehydrogenase combined with the deletion of apyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate carboxykinase combined with theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a pyruvate carboxylase gene and aphosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvate kinasegene and a pyruvate carboxylase gene and a phosphoenolpyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of a phosphoenolpyruvate carboxykinase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene and a pyruvate carboxylase gene and aphosphoenolpyruvate carboxylase gene, the overexpression of anoxaloacetate decarboxylase combined with the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a pyruvate carboxylase gene and aphosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and pyruvate carboxylase gene and a phosphoenolpyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase and the overexpression of a malate dehydrogenase combinedwith the deletion of a pyruvate kinase gene and a pyruvate carboxylasegene and a phosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of anoxaloacetate decarboxylase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene.

According to another preferred embodiment of the method and/or cell ofthe disclosure, the cell comprises a modification for reduced productionof acetate compared to a non-modified progenitor. The modification canbe any one or more chosen from the group comprising overexpression of anacetyl-coenzyme A synthetase, a full or partial knock-out or renderedless functional pyruvate dehydrogenase and a full or partial knock-outor rendered less functional lactate dehydrogenase.

In a further embodiment of the method and/or cell of the disclosure, thecell is modified in the expression or activity of at least oneacetyl-coenzyme A synthetase like e.g., acs from E. coli, S. cerevisiae,H. sapiens, M. musculus. In a preferred embodiment, the acetyl-coenzymeA synthetase is an endogenous protein of the cell with a modifiedexpression or activity, preferably the endogenous acetyl-coenzyme Asynthetase is overexpressed; alternatively, the acetyl-coenzyme Asynthetase is a heterologous protein that is heterogeneously introducedand expressed in the cell, preferably overexpressed. The endogenousacetyl-coenzyme A synthetase can have a modified expression in the cellwhich also expresses a heterologous acetyl-coenzyme A synthetase. In amore preferred embodiment, the cell is modified in the expression oractivity of the acetyl-coenzyme A synthetase acs from E. coli (UniProtID P27550). In another and/or additional preferred embodiment, the cellis modified in the expression or activity of a functional homolog,variant or derivative of acs from E. coli (UniProt ID P27550) having atleast 80% overall sequence identity to the full-length of thepolypeptide from E. coli (UniProt ID P27550) and having acetyl-coenzymeA synthetase activity.

In an alternative and/or additional further embodiment of the methodand/or cell of the disclosure, the cell is modified in the expression oractivity of at least one pyruvate dehydrogenase like e.g., from E. coli,S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment,the cell has been modified to have at least one partially or fullyknocked out or mutated pyruvate dehydrogenase encoding gene by meansgenerally known by the person skilled in the art resulting in at leastone protein with less functional or being disabled for pyruvatedehydrogenase activity. In a more preferred embodiment, the cell has afull knock-out in the poxB encoding gene resulting in a cell lackingpyruvate dehydrogenase activity.

In an alternative and/or additional further embodiment of the methodand/or cell of the disclosure, the cell is modified in the expression oractivity of at least one lactate dehydrogenase like e.g., from E. coli,S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment,the cell has been modified to have at least one partially or fullyknocked out or mutated lactate dehydrogenase encoding gene by meansgenerally known by the person skilled in the art resulting in at leastone protein with less functional or being disabled for lactatedehydrogenase activity. In a more preferred embodiment, the cell has afull knock-out in the ldhA encoding gene resulting in a cell lackinglactate dehydrogenase activity.

According to another preferred embodiment of the method and/or cell ofthe disclosure, the cell comprises a lower or reduced expression and/orabolished, impaired, reduced or delayed activity of any one or more ofthe proteins comprising beta-galactosidase, galactosideO-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase,glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor,ribonucleotide monophosphatase, EIICBA-Nag,UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase,L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase,N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase,EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphateuridylyltransferase, glucose-1-phosphate adenylyltransferase,glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1,ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphateisomerase, aerobic respiration control protein, transcriptionalrepressor IclR, lon protease, glucose-specific translocatingphosphotransferase enzyme IIBC component ptsG, glucose-specifictranslocating phosphotransferase (PTS) enzyme IIBC component malX,enzyme IIAGlc, beta-glucoside specific PTS enzyme II, fructose-specificPTS multiphosphoryl transfer protein FruA and FruB, ethanoldehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetatekinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvatedecarboxylase compared to a non-modified progenitor.

According to another preferred embodiment of the method and/or cell ofthe disclosure, the cell comprises a catabolic pathway for selectedmono-, di- or oligosaccharides which is at least partially inactivated,the mono-, di-, or oligosaccharides being involved in and/or requiredfor the production of a di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end.

According to another preferred embodiment of the method and/or cell ofthe disclosure, the cell is using a precursor for the production of adi- or oligosaccharide having an N-acetylglucosamine unit at thereducing end, preferably the precursor being fed to the cell from thecultivation medium. According to a more preferred aspect of the methodand/or cell, the cell is using at least two precursors for theproduction of the di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end, preferably the precursors being fed to thecell from the cultivation medium. According to another preferred aspectof the method and/or cell of the disclosure, the cell is producing atleast one precursor, preferably at least two precursors, for theproduction of the di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end. In a preferred embodiment of the method and/orcell, the precursor that is used by the cell for the production of a di-or oligosaccharide having an N-acetylglucosamine unit at the reducingend is completely converted into the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end.

According to another preferred embodiment of the method and/or cell ofthe disclosure, the cell produces 90 g/L or more of a di- oroligosaccharide having an N-acetylglucosamine unit at the reducing endin the whole broth and/or supernatant. In a more preferred embodiment,the di- or oligosaccharide having an N-acetylglucosamine unit at thereducing end produced in the whole broth and/or supernatant has a purityof at least 80% measured on the total amount of di- or oligosaccharidehaving an N-acetylglucosamine unit at the reducing end and its precursorproduced by the cell in the whole broth and/or supernatant,respectively.

Another embodiment of the disclosure provides for a method and a cellwherein a di- or oligosaccharide with a GlcNAc unit at the reducing endis produced in and/or by a fungal, yeast, bacterial, insect, animal,plant, or protozoan cell as described herein. The cell is chosen fromthe list comprising a bacterium, a yeast, a protozoan or a fungus, or,refers to a plant or animal cell. The latter bacterium preferablybelongs to the phylum of the Proteobacteria or the phylum of theFirmicutes or the phylum of the Cyanobacteria or the phylumDeinococcus-Thermus. The latter bacterium belonging to the phylumProteobacteria belongs preferably to the family Enterobacteriaceae,preferably to the species Escherichia coli. The latter bacteriumpreferably relates to any strain belonging to the species Escherichiacoli such as but not limited to Escherichia coli B, Escherichia coli C,Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. Morespecifically, the latter term relates to cultivated Escherichia colistrains—designated as E. coli K12 strains—which are well-adapted to thelaboratory environment, and, unlike wild type strains, have lost theirability to thrive in the intestine. Well-known examples of the E. coliK12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060,MC1061, MC4100, JM101, NZN111 and AA200. Hence, the disclosurespecifically relates to a mutated and/or transformed Escherichia colicell or strain as indicated above wherein the E. coli strain is a K12strain. More preferably, the Escherichia coli K12 strain is E. coliMG1655. The latter bacterium belonging to the phylum Firmicutes belongspreferably to the Bacilli, preferably Lactobacilliales, with memberssuch as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacillaleswith members such as from the genus Bacillus, such as Bacillus subtilisor, B. amyloliquefaciens. The latter Bacterium belonging to the phylumActinobacteria, preferably belonging to the family of theCorynebacteriaceae, with members Corynebacterium glutamicum or C.afermentans, or belonging to the family of the Streptomycetaceae withmembers Streptomyces griseus or S. fradiae. The latter yeast preferablybelongs to the phylum of the Ascomycota or the phylum of theBasidiomycota or the phylum of the Deuteromycota or the phylum of theZygomycetes. The latter yeast belongs preferably to the genusSaccharomyces (with members like e.g., Saccharomyces cerevisiae, S.bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members likee.g., Pichia pastoris, P. anomala, P. kluyveri), Komagataella,Hansenula, Kluyveromyces (with members like e.g., Kluyveromyces lactis,K. marxianus, K thermotolerans), Debaromyces, Yarrowia (like e.g.,Yarrowia lipolytica) or Starmerella (like e.g., Starmerella bombicola).The latter yeast is preferably selected from Pichia pastoris, Yarrowiahpolitica, Saccharomyces cerevisiae and Kluyveromyces lactis. The latterfungus belongs preferably to the genus Rhizopus, Dictyostelium,Penicillium, Mucor or Aspergillus. Plant cells include cells offlowering and non-flowering plants, as well as algal cells, for example,Chlamydomonas, Chlorella, etc. Preferably, the plant is a tobacco,alfalfa, rice, tomato, cotton, rapeseed, soy, maize or corn plant. Thelatter animal cell is preferably derived from non-human mammals (e.g.,cattle, buffalo, pig, sheep, mouse, rat), birds (e.g., chicken, duck,ostrich, turkey, pheasant), fish (e.g., swordfish, salmon, tuna, seabass, trout, catfish), invertebrates (e.g., lobster, crab, shrimp,clams, oyster, mussel, sea urchin), reptiles (e.g., snake, alligator,turtle), amphibians (e.g., frogs) or insects (e.g., fly, nematode) or isa genetically modified cell line derived from human cells excludingembryonic stem cells. Both human and non-human mammalian cells arepreferably chosen from the list comprising an epithelial cell like e.g.,a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293 or HEK293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO)cell, a murine myeloma cell like e.g., an N20, SP2/0 or YB2/0 cell, anNIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof suchas described in WO 2021067641. The latter insect cell is preferablyderived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyxmori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cellsor Drosophila melanogaster like e.g., Drosophila S2 cells. The latterprotozoan cell preferably is a Leishmania tarentolae cell.

In a preferred embodiment of the method and/or cell of the disclosure,the cell is a viable Gram-negative bacterium that comprises a reduced orabolished synthesis of poly-N-acetyl-glucosamine (PNAG), EnterobacterialCommon Antigen (ECA), cellulose, colanic acid, core oligosaccharides,Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan,and/or trehalose compared to a non-modified progenitor.

In a more preferred embodiment of the method and/or cell, the reduced orabolished synthesis of poly-N-acetyl-glucosamine (PNAG), EnterobacterialCommon Antigen (ECA), cellulose, colanic acid, core oligosaccharides,Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan,and/or trehalose is provided by a mutation in any one or moreglycosyltransferases involved in the synthesis of any one of thepoly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA),cellulose, colanic acid, core oligosaccharides, OsmoregulatedPeriplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose,wherein the mutation provides for a deletion or lower expression of anyone of the glycosyltransferases. The glycosyltransferases compriseglycosyltransferase genes encoding poly-N-acetyl-D-glucosamine synthasesubunits, UDP-N-acetylglucosamine-undecaprenyl-phosphateN-acetylglucosaminephosphotransferase, Fuc4NAc(4-acetamido-4,6-dideoxy-D-galactose) transferase,UDP-N-acetyl-D-mannosaminuronic acid transferase, theglycosyltransferase genes encoding the cellulose synthase catalyticsubunits, the cellulose biosynthesis protein, colanic acid biosynthesisglucuronosyltransferase, colanic acid biosynthesisgalactosyltransferase, colanic acid biosynthesis fucosyltransferase,UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase,putative colanic biosynthesis glycosyl transferase,UDP-glucuronate:LPS(HepIII) glycosyltransferase, ADP-heptose-LPSheptosyltransferase 2, ADP-heptose:LPS heptosyltransferase 1, putativeADP-heptose:LPS heptosyltransferase 4, lipopolysaccharide corebiosynthesis protein, UDP-glucose:(glucosyl)LPSα-1,2-glucosyltransferase, UDP-D-glucose: (glucosyl)LPSα-1,3-glucosyltransferase, UDP-D-galactose:(glucosyl)lipopolysaccharide-1,6-D-galactosyltransferase,lipopolysaccharide glucosyltransferase I, lipopolysaccharide coreheptosyltransferase 3, β-1,6-galactofuranosyltransferase,undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase,lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bactoprenol glucosyltransferase, putative family 2 glycosyltransferase, the osmoregulatedperiplasmic glucans (OPG) biosynthesis protein G, OPG biosynthesisprotein H, glucosylglycerate phosphorylase, glycogen synthase,1,4-α-glucan branching enzyme, 4-α-glucanotransferase andtrehalose-6-phosphate synthase. In an exemplary embodiment, the cell ismutated in any one or more of the glycosyltransferases comprising pgaC,pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ,wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ,wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsAand yaiP, wherein the mutation provides for a deletion or lowerexpression of any one of the glycosyltransferases.

In an alternative and/or additional preferred embodiment of the methodand/or cell, the reduced or abolished synthesis ofpoly-N-acetyl-glucosamine (PNAG) is provided by over-expression of acarbon storage regulator encoding gene, deletion of a Na+/H+ antiporterregulator encoding gene and/or deletion of the sensor histidine kinaseencoding gene.

The microorganism or cell as described herein is capable to grow on amonosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol,glycerol, a complex medium including molasses, corn steep liquor,peptone, tryptone, yeast extract or a mixture thereof like e.g., a mixedfeedstock, preferably a mixed monosaccharide feedstock like e.g.,hydrolysed sucrose, as the main carbon source. With the term “complexmedium” is meant a medium for which the exact constitution is notdetermined. With the term main is meant the most important carbon sourcefor the bioproducts of interest, biomass formation, carbon dioxideand/or by-products formation (such as acids and/or alcohols, such asacetate, lactate, and/or ethanol), i.e., 20, 30, 40, 50, 60, 70, 75, 80,85, 90, 95, 98, 99% of all the required carbon is derived from theabove-indicated carbon source. In an embodiment of the disclosure, thecarbon source is the sole carbon source for the organism, i.e., 100% ofall the required carbon is derived from the above-indicated carbonsource. Common main carbon sources comprise but are not limited toglucose, glycerol, fructose, maltose, lactose, arabinose,malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose,sucrose, galactose, mannose, methanol, ethanol, trehalose, starch,cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructosesyrup, acetate, citrate, lactate and pyruvate. With the term complexmedium is meant a medium for which the exact constitution is notdetermined. Examples are molasses, corn steep liquor, peptone, tryptoneor yeast extract.

In a further preferred embodiment, the microorganism or cell describedherein is using a split metabolism having a production pathway and abiomass pathway as described in WO 2012/007481, which is hereinincorporated by reference. The organism can, for example, be geneticallymodified to accumulate fructose-6-phosphate by altering the genesselected from the phosphoglucoisomerase gene, phosphofructokinase gene,fructose-6-phosphate aldolase gene, fructose isomerase gene, and/orfructose:PEP phosphotransferase gene.

According to another embodiment of the method of the disclosure, theconditions permissive to produce the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end comprise the use of aculture medium comprising at least one precursor and/or acceptor for theproduction of the a di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end. Preferably, the culture medium contains atleast one precursor selected from the group comprising lactose,galactose, fucose, sialic acid.

According to an alternative and/or additional embodiment of the methodof the disclosure, the conditions permissive to produce the di- oroligosaccharide having an N-acetylglucosamine unit at the reducing endcomprise adding to the culture medium at least one precursor and/oracceptor feed for the production of the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end.

According to an alternative embodiment of the method of the disclosure,the conditions permissive to produce the di- or oligosaccharide havingan N-acetylglucosamine unit at the reducing end comprise the use of aculture medium to cultivate a cell of disclosure for the production of adi- or oligosaccharide having an N-acetylglucosamine unit at thereducing end wherein the culture medium lacks any precursor and/oracceptor for the production of the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end and is combined with afurther addition to the culture medium of at least one precursor and/oracceptor feed for the production of the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end.

In a preferred embodiment, the method for the production of a di- oroligosaccharide having an N-acetylglucosamine unit at the reducing endas described herein comprises at least one of the following steps:

-   -   i) Use of a culture medium comprising at least one precursor        and/or acceptor;    -   ii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed wherein the total reactor volume        ranges from 250 mL (milliliter) to 10.000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        2-fold of the volume of the culture medium before the addition        of the precursor and/or acceptor feed;    -   iii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed wherein the total reactor volume        ranges from 250 mL (milliliter) to 10.000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        2-fold of the volume of the culture medium before the addition        of the precursor and/or acceptor feed and wherein preferably,        the pH of the precursor and/or acceptor feed is set between 3        and 7 and wherein preferably, the temperature of the precursor        and/or acceptor feed is kept between 20° C. and 80° C.;    -   iv) Adding at least one precursor and/or acceptor feed in a        continuous manner to the culture medium over the course of 1        day, 2 days, 3 days, 4 days, 5 days by means of a feeding        solution;    -   v) Adding at least one precursor and/or acceptor feed in a        continuous manner to the culture medium over the course of 1        day, 2 days, 3 days, 4 days, 5 days by means of a feeding        solution and wherein preferably, the pH of the feeding solution        is set between 3 and 7 and wherein preferably, the temperature        of the feeding solution is kept between 20° C. and 80° C.;

the method resulting in a di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end with a concentration of atleast 50 g/L, preferably at least 75 g/L, more preferably at least 90g/L, more preferably at least 100 g/L, more preferably at least 125 g/L,more preferably at least 150 g/L, more preferably at least 175 g/L, morepreferably at least 200 g/L in the final cultivation.

In another and/or additional preferred embodiment, the method for theproduction of a di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end as described herein comprises at least one ofthe following steps:

-   -   i) Use of a culture medium comprising at least 50, more        preferably at least 75, more preferably at least 100, more        preferably at least 120, more preferably at least 150 gram of        lactose per liter of initial reactor volume wherein the reactor        volume ranges from 250 mL to 10.000 m³ (cubic meter);    -   ii) Adding to the culture medium at least one precursor and/or        acceptor in one pulse or in a discontinuous (pulsed) manner        wherein the total reactor volume ranges from 250 mL (milliliter)        to 10.000 m³ (cubic meter), preferably so that the final volume        of the culture medium is not more than three-fold, preferably        not more than two-fold, more preferably less than 2-fold of the        volume of the culture medium before the addition of the        precursor and/or acceptor feed pulse(s);    -   iii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed in one pulse or in a        discontinuous (pulsed) manner wherein the total reactor volume        ranges from 250 mL (milliliter) to 10.000 m³ (cubic meter),        preferably so that the final volume of the culture medium is not        more than three-fold, preferably not more than two-fold, more        preferably less than 2-fold of the volume of the culture medium        before the addition of the precursor and/or acceptor feed        pulse(s) and wherein preferably, the pH of the precursor and/or        acceptor feed pulse(s) is set between 3 and 7 and wherein        preferably, the temperature of the precursor and/or acceptor        feed pulse(s) is kept between 20° C. and 80° C.;    -   iv) Adding at least one precursor and/or acceptor feed in a        discontinuous (pulsed) manner to the culture medium over the        course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4        hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days        by means of a feeding solution;    -   v) Adding at least one precursor and/or acceptor feed in a        discontinuous (pulsed) manner to the culture medium over the        course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4        hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days        by means of a feeding solution and wherein preferably, the pH of        the feeding solution is set between 3 and 7 and wherein        preferably, the temperature of the feeding solution is kept        between 20° C. and 80° C.;

the method resulting in a di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end with a concentration of atleast 50 g/L, preferably at least 75 g/L, more preferably at least 90g/L, more preferably at least 100 g/L, more preferably at least 125 g/L,more preferably at least 150 g/L, more preferably at least 175 g/L, morepreferably at least 200 g/L in the final cultivation.

In a further, more preferred embodiment, the method for the productionof a di- or oligosaccharide having an N-acetylglucosamine unit at thereducing end as described herein comprises at least one of the followingsteps:

-   -   i) Use of a culture medium comprising at least 50, more        preferably at least 75, more preferably at least 100, more        preferably at least 120, more preferably at least 150 gram of        lactose per liter of initial reactor volume wherein the reactor        volume ranges from 250 mL to 10.000 m³ (cubic meter);    -   ii) Adding to the culture medium a lactose feed comprising at        least 50, more preferably at least 75, more preferably at least        100, more preferably at least 120, more preferably at least 150        gram of lactose per liter of initial reactor volume wherein the        total reactor volume ranges from 250 mL (milliliter) to 10.000        m³ (cubic meter), preferably in a continuous manner, and        preferably so that the final volume of the culture medium is not        more than three-fold, preferably not more than two-fold, more        preferably less than 2-fold of the volume of the culture medium        before the addition of the lactose feed;    -   iii) Adding to the culture medium a lactose feed comprising at        least 50, more preferably at least 75, more preferably at least        100, more preferably at least 120, more preferably at least 150        gram of lactose per liter of initial reactor volume wherein the        reactor volume ranges from 250 mL to 10.000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        2-fold of the volume of the culture medium before the addition        of the lactose feed and wherein preferably the pH of the lactose        feed is set between 3 and 7 and wherein preferably the        temperature of the lactose feed is kept between 20° C. and 80°        C.;    -   iv) Adding a lactose feed in a continuous manner to the culture        medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days        by means of a feeding solution;    -   v) Adding a lactose feed in a continuous manner to the culture        medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days        by means of a feeding solution and wherein the concentration of        the lactose feeding solution is 50 g/L, preferably 75 g/L, more        preferably 100 g/L, more preferably 125 g/L, more preferably 150        g/L, more preferably 175 g/L, more preferably 200 g/L, more        preferably 225 g/L, more preferably 250 g/L, more preferably 275        g/L, more preferably 300 g/L, more preferably 325 g/L, more        preferably 350 g/L, more preferably 375 g/L, more preferably,        400 g/L, more preferably 450 g/L, more preferably 500 g/L, even        more preferably, 550 g/L, most preferably 600 g/L; and wherein        preferably the pH of the feeding solution is set between 3 and 7        and wherein preferably the temperature of the feeding solution        is kept between 20° C. and 80° C.;

the method resulting in a di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end with a concentration of atleast 50 g/L, preferably at least 75 g/L, more preferably at least 90g/L, more preferably at least 100 g/L, more preferably at least 125 g/L,more preferably at least 150 g/L, more preferably at least 175 g/L, morepreferably at least 200 g/L in the final cultivation.

In a further embodiment of the methods described herein the host cellsare cultivated for at least about 60, 80, 100, or about 120 hours or ina continuous manner.

In a preferred embodiment, a carbon source is provided, preferablysucrose, in the culture medium for 3 or more days, preferably up to 7days; and/or provided, in the culture medium, at least 100,advantageously at least 105, more advantageously at least 110, even moreadvantageously at least 120 grams of sucrose per liter of initialculture volume in a continuous manner, so that the final volume of theculture medium is not more than three-fold, advantageously not more thantwo-fold, more advantageously less than two-fold of the volume of theculturing medium before the culturing.

Preferably, when performing the method as described herein, a firstphase of exponential cell growth is provided by adding a carbon source,preferably glucose or sucrose, to the culture medium before theprecursor is added to the cultivation in a second phase.

In another preferred embodiment of the method of disclosure, a firstphase of exponential cell growth is provided by adding a carbon-basedsubstrate, preferably glucose or sucrose, to the culture mediumcomprising a precursor, followed by a second phase wherein only acarbon-based substrate, preferably glucose or sucrose, is added to thecultivation.

In another preferred embodiment of the method of disclosure, a firstphase of exponential cell growth is provided by adding a carbon-basedsubstrate, preferably glucose or sucrose, to the culture mediumcomprising a precursor, followed by a second phase wherein acarbon-based substrate, preferably glucose or sucrose, and a precursorare added to the cultivation.

In an alternative preferable embodiment, in the method as describedherein, the precursor is added already in the first phase of exponentialgrowth together with the carbon-based substrate.

The method for producing a di- or oligosaccharide with a GlcNAc unit atthe reducing end as described herein, further comprises a step ofseparating the di- or oligosaccharide with a GlcNAc unit at the reducingend from the cell or the medium of its growth.

The term “separating” means harvesting, collecting, or retrieving thedi- or oligosaccharide with a GlcNAc unit at the reducing end or amixture thereof) from the cell and/or the medium of its growth.

The di- or oligosaccharide with a GlcNAc unit at the reducing end can beseparated in a conventional manner from the aqueous culture medium, inwhich the cell was grown. In case the di- or oligosaccharide with aGlcNAc unit at the reducing end is still present in the cells producingthe di- or oligosaccharide with a GlcNAc unit at the reducing end,conventional manners to free or to extract the di- or oligosaccharidewith a GlcNAc unit at the reducing end out of the cells can be used,such as cell destruction using high pH, heat shock, sonication, Frenchpress, homogenization, enzymatic hydrolysis, chemical hydrolysis,solvent hydrolysis, detergent, hydrolysis, . . . . The culture mediumand/or cell extract together and separately can then be further used forseparating the di- or oligosaccharide with a GlcNAc unit at the reducingend. This preferably involves clarifying the di- or oligosaccharide witha GlcNAc unit at the reducing end containing mixture to remove suspendedparticulates and contaminants, particularly cells, cell components,insoluble metabolites and debris produced by culturing the geneticallymodified cell. In this step, the di- or oligosaccharide with a GlcNAcunit at the reducing end containing mixture can be clarified in aconventional manner. Preferably, the di- or oligosaccharide with aGlcNAc unit at the reducing end containing mixture is clarified bycentrifugation, flocculation, decantation and/or filtration. Anotherstep of separating the di- or oligosaccharide with a GlcNAc unit at thereducing end from the di- or oligosaccharide with a GlcNAc unit at thereducing end containing mixture preferably involves removingsubstantially all the proteins, as well as peptides, amino acids, RNAand DNA and any endotoxins and glycolipids that could interfere with thesubsequent separation step, from the di- or oligosaccharide with aGlcNAc unit at the reducing end containing mixture, preferably after ithas been clarified. In this step, proteins and related impurities can beremoved from the di- or oligosaccharide with a GlcNAc unit at thereducing end containing mixture in a conventional manner. Preferably,proteins, salts, by-products, color, endotoxins and other relatedimpurities are removed from the di- or oligosaccharide with a GlcNAcunit at the reducing end containing mixture by ultrafiltration,nanofiltration, two-phase partitioning, reverse osmosis,microfiltration, activated charcoal or carbon treatment, treatment withnon-ionic surfactants, enzymatic digestion, tangential flowhigh-performance filtration, tangential flow ultrafiltration,electrophoresis (e.g., using slab-polyacrylamide or sodium dodecylsulphate-polyacrylamide gel electrophoresis (PAGE)), affinitychromatography (using affinity ligands including e.g., DEAE-SEPHAROSE®,poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices),ion exchange chromatography (such as but not limited to cation exchange,anion exchange, mixed bed ion exchange, inside-out ligand attachment),hydrophobic interaction chromatography and/or gel filtration (i.e., sizeexclusion chromatography), particularly by chromatography, moreparticularly by ion exchange chromatography or hydrophobic interactionchromatography or ligand exchange chromatography. With the exception ofsize exclusion chromatography, proteins and related impurities areretained by a chromatography medium or a selected membrane, while thedi- or oligosaccharide with a GlcNAc unit at the reducing end remains inthe di- or oligosaccharide with a GlcNAc unit at the reducing endcontaining mixture.

In a further preferred embodiment, the methods as described herein alsoprovide for a further purification of the di- or oligosaccharide with aGlcNAc unit at the reducing end. A further purification of the di- oroligosaccharide with a GlcNAc unit at the reducing end may beaccomplished, for example, by use of (activated) charcoal or carbon,nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment orion exchange to remove any remaining DNA, protein, LPS, endotoxins, orother impurity. Alcohols, such as ethanol, and aqueous alcohol mixturescan also be used. Another purification step is accomplished bycrystallization, evaporation or precipitation of the product. Anotherpurification step is to dry, e.g., spray dry, lyophilize, spray freezedry, freeze spray dry, band dry, belt dry, vacuum band dry, vacuum beltdry, drum dry, roller dry, vacuum drum dry or vacuum roller dry theproduced di- or oligosaccharide with GlcNAc at the reducing end.

In an exemplary embodiment, the separation and purification of theproduced di- or oligosaccharide having an N-acetylglucosamine unit atthe reducing end is made in a process, comprising the following steps inany order:

-   -   a) contacting the cultivation or a clarified version thereof        with a nanofiltration membrane with a molecular weight cut-off        (MWCO) of 600-3500 Da ensuring the retention of produced di- or        oligosaccharide and allowing at least a part of the proteins,        salts, by-products, color and other related impurities to pass,    -   b) conducting a diafiltration process on the retentate from step        a), using the membrane, with an aqueous solution of an inorganic        electrolyte, followed by optional diafiltration with pure water        to remove excess of the electrolyte,    -   c) and collecting the retentate enriched in the di- or        oligosaccharide having an N-acetylglucosamine unit at the        reducing end in the form of a salt from the cation of the        electrolyte,    -   d) preferably the retentate is dried.

In an alternative exemplary embodiment, the separation and purificationof the produced di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end is made in a process, comprising the followingsteps in any order: subjecting the cultivation or a clarified versionthereof to two membrane filtration steps using different membranes,wherein—one membrane has a molecular weight cut-off of between about 300Dalton to about 500 Dalton, and—the other membrane as a molecular weightcut-off of between about 600 Dalton to about 800 Dalton.

In an alternative exemplary embodiment, the separation and purificationof the produced di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end is made in a process, comprising the followingsteps in any order comprising the step of treating the cultivation or aclarified version thereof with a strong cation exchange resin in H+-formand a weak anion exchange resin in free base form.

In an alternative exemplary embodiment, the separation and purificationof the produced di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end is made in the following way. The cultivationcomprising the produced di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end, biomass, medium componentsand contaminants is applied to the following separation and purificationsteps:

-   -   i) separation of biomass from the cultivation,    -   ii) cationic ion exchanger treatment for the removal of        positively charged material,    -   iii) anionic ion exchanger treatment for the removal of        negatively charged material,    -   iv) nanofiltration step and/or electrodialysis step,

wherein a purified solution comprising the produced di- oroligosaccharide having an N-acetylglucosamine unit at the reducing endat a purity of greater than or equal to 80 percent is provided.Optionally the purified solution is dried by any one or more dryingsteps chosen from the list comprising spray drying, lyophilization,spray freeze drying, freeze spray drying, band drying, belt drying,vacuum band drying, vacuum belt drying, drum drying, roller drying,vacuum drum drying and vacuum roller drying.

In an alternative exemplary embodiment, the separation and purificationof the produced di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end is made in a process, comprising the followingsteps in any order: enzymatic treatment of the cultivation; removal ofthe biomass from the cultivation; ultrafiltration; nanofiltration; and acolumn chromatography step. Preferably such column chromatography is asingle column or a multiple column. Further preferably the columnchromatography step is simulated moving bed chromatography. Suchsimulated moving bed chromatography preferably comprises i) at least 4columns, wherein at least one column comprises a weak or strong cationexchange resin; and/or ii) four zones I, II, III and IV with differentflow rates; and/or iii) an eluent comprising water; and/or iv) anoperating temperature of 15 degrees to 60 degrees centigrade.Preferably, the process further comprises a step of drying chosen fromthe list comprising spray drying, lyophilization, spray freeze drying,freeze spray drying, band drying, belt drying, vacuum band drying,vacuum belt drying, drum drying, roller drying, vacuum drum drying andvacuum roller drying.

In a specific embodiment, the disclosure provides the produced di- oroligosaccharide with GlcNAc at the reducing end which is dried to powderby any one or more drying steps chosen from the list comprising spraydrying, lyophilization, spray freeze drying, freeze spray drying, banddrying, belt drying, vacuum band drying, vacuum belt drying, drumdrying, roller drying, vacuum drum drying and vacuum roller drying,wherein the dried powder contains <15 percent-wt. of water, preferably<10 percent-wt. of water, more preferably <7 percent-wt. of water, mostpreferably <5 percent-wt. of water.

In a third aspect, the disclosure provides for the use of ametabolically engineered cell as described herein for the production ofa di- or oligosaccharide having an N-acetylglucosamine unit at thereducing end, preferably for the production of LNB or LacNAc, morepreferably for the production of an oligosaccharide containing LNB orLacNAc at the reducing end, even more preferably for the production ofsialylated and/or fucosylated and/or galactosylated and/orGlcNAc-modified forms of LNB or LacNAc, or most preferably for theproduction of sialylated and/or fucosylated and/or galactosylated and/orGlcNAc-modified forms of an oligosaccharide containing LNB or LacNAc atthe reducing end.

In another preferred embodiment of the third aspect, a metabolicallyengineered cell as described herein is used for the production of aneutral di- or oligosaccharide having an N-acetylglucosamine unit at thereducing end, more preferably a neutral oligosaccharide containing LNBor LacNAc at the reducing end.

In another preferred embodiment of the third aspect, a metabolicallyengineered cell as described herein is used for the production of amixture of di- and/or oligosaccharides having an N-acetylglucosamine(GlcNAc) unit at the reducing end as described herein.

In another preferred embodiment of the third aspect, a metabolicallyengineered cell as described herein is used for the production of amixture comprising (i) a di- or oligosaccharide havingN-acetylglucosamine (GlcNAc) unit at the reducing end (or a mixturethereof as disclosed herein) and (ii) one or more lactose-basedmammalian milk oligosaccharides, preferably one or more lactose-basedhuman milk oligosaccharides as described herein.

For identification of the di- or oligosaccharide with a GlcNAc unit atthe reducing end produced in the cell as described herein, the monomericbuilding blocks (e.g., the monosaccharide or glycan unit composition),the anomeric configuration of side chains, the presence and location ofsubstituent groups, degree of polymerization/molecular weight and thelinkage pattern can be identified by standard methods known in the art,such as, e.g., methylation analysis, reductive cleavage, hydrolysis,GC-MS (gas chromatography-mass spectrometry), MALDI-MS (Matrix-assistedlaser desorption/ionization-mass spectrometry), ESI-MS (Electrosprayionization-mass spectrometry), HPLC (High-Performance Liquidchromatography with ultraviolet or refractive index detection),HPAEC-PAD (High-Performance Anion-Exchange chromatography with PulsedAmperometric Detection), CE (capillary electrophoresis), IR(infrared)/Raman spectroscopy, and NMR (Nuclear magnetic resonance)spectroscopy techniques. The crystal structure can be solved using,e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy),and WAXS (wide-angle X-ray scattering). The degree of polymerization(DP), the DP distribution, and polydispersity can be determined by,e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusionchromatography). To identify the monomeric components of the saccharidemethods such as, e.g., acid-catalyzed hydrolysis, HPLC (high performanceliquid chromatography) or GLC (gas-liquid chromatography) (afterconversion to alditol acetates) may be used. To determine the glycosidiclinkages, the saccharide is methylated with methyl iodide and strongbase in DMSO, hydrolysis is performed, a reduction to partiallymethylated alditols is achieved, an acetylation to methylated alditolacetates is performed, and the analysis is carried out by GLC/MS(gas-liquid chromatography coupled with mass spectrometry). To determinethe oligosaccharide sequence, a partial depolymerization is carried outusing an acid or enzymes to determine the structures. To identify theanomeric configuration, the oligosaccharide is subjected to enzymaticanalysis, e.g., it is contacted with an enzyme that is specific for aparticular type of linkage, e.g., beta-galactosidase, oralpha-glucosidase, etc., and NMR may be used to analyze the products.

Products comprising the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end.

In some embodiments, an di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end (or a mixture as describedherein) produced as described herein is incorporated into a food (e.g.,human food or feed), dietary supplement, pharmaceutical ingredient,cosmetic ingredient or medicine. In some embodiments, the di- oroligosaccharide having an N-acetylglucosamine unit at the reducing endis mixed with one or more ingredients suitable for food, feed, dietarysupplement, pharmaceutical ingredient, cosmetic ingredient or medicine.

In some embodiments, the dietary supplement comprises at least oneprebiotic ingredient and/or at least one probiotic ingredient.

A “prebiotic” is a substance that promotes growth of microorganismsbeneficial to the host, particularly microorganisms in thegastrointestinal tract. In some embodiments, a dietary supplementprovides multiple prebiotics, including the di- or oligosaccharidehaving an N-acetylglucosamine unit at the reducing end produced and/orpurified by a process disclosed in this specification, to promote growthof one or more beneficial microorganisms. Examples of prebioticingredients for dietary supplements include other prebiotic molecules(such as HMOs) and plant polysaccharides (such as inulin, pectin,b-glucan and xylooligosaccharide). A “probiotic” product typicallycontains live microorganisms that replace or add to gastrointestinalmicroflora, to the benefit of the recipient. Examples of suchmicroorganisms include Lactobacillus species (for example, L.acidophilus and L. bulgaricus), Bifidobacterium species (for example, B.animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomycesboulardii. In some embodiments, an di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end produced and/or purified bya process of this specification is orally administered in combinationwith such microorganism.

Examples of further ingredients for dietary supplements includedisaccharides (such as lactose), monosaccharides (such as glucose andgalactose), thickeners (such as gum arabic), acidity regulators (such astrisodium citrate), water, skimmed milk, and flavourings.

In some embodiments, the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end (or a mixture as describedherein) is incorporated into a human baby food (e.g., infant formula).Infant formula is generally a manufactured food for feeding to infantsas a complete or partial substitute for human breast milk. In someembodiments, infant formula is sold as a powder and prepared for bottle-or cup-feeding to an infant by mixing with water. The composition ofinfant formula is typically designed to be roughly mimic human breastmilk. In some embodiments, an di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end (or a mixture as describedherein) produced and/or purified by a process in this specification isincluded in infant formula to provide nutritional benefits similar tothose provided by the oligosaccharides in human breast milk. In someembodiments, the di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end is mixed with one or more ingredients of theinfant formula. Examples of infant formula ingredients include non-fatmilk, carbohydrate sources (e.g., lactose), protein sources (e.g., wheyprotein concentrate and casein), fat sources (e.g., vegetable oils—suchas palm, high oleic safflower oil, rapeseed, coconut and/or sunfloweroil; and fish oils), vitamins (such as vitamins A, B6, B12, C and D),minerals (such as potassium citrate, calcium citrate, magnesiumchloride, sodium chloride, sodium citrate and calcium phosphate) andpossibly human milk oligosaccharides (HMOs). Such HMOs may include, forexample, DiFL, lacto-N-triose II, LNT, LNnT, lacto-N-fucopentaose I,lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaoseIII, lacto-N-fucopentaose V, lacto-N-neofucopentaose V,lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6′-galactosyllactose,3′-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose.

In some embodiments, the one or more infant formula ingredients comprisenon-fat milk, a carbohydrate source, a protein source, a fat source,and/or a vitamin and mineral.

In some embodiments, the one or more infant formula ingredients compriselactose, whey protein concentrate and/or high oleic safflower oil.

In some embodiments, the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end (or a mixture as describedherein) concentration in the infant formula is approximately the sameconcentration as the oligosaccharide's concentration generally presentin human breast milk. In some embodiments, the concentration of di- oroligosaccharide in the infant formula is approximately the sameconcentration as the concentration of that oligosaccharide generallypresent in human breast milk.

In some embodiments, the di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end (or a mixture as describedherein) is incorporated into a feed preparation, wherein the feed ischosen from the list comprising petfood, animal milk replacer,veterinary product, post weaning feed, or creep feed.

In some embodiments, a food, a feed, a dietary supplement, apharmaceutical ingredient and/or a medicine comprises at least oneimmunomodulatory ingredient.

“An immunomodulatory” ingredient is a substance that modifies the immuneresponse or the functioning of the immune system. An “immunomodulatory”ingredient can modify the immune response by an increase(immunostimulators) or a decrease (immunosuppressives) of the productionof serum antibodies. Immunostimulators are used, among others, toenhance the immune response against infectious diseases, tumours,primary or secondary immunodeficiency, and alterations in antibodytransfer. Immunosuppressive ingredients are used to reduce the immuneresponse against transplanted organs and to treat autoimmune diseasessuch as pemphigus, lupus, or allergies. In some embodiments, theimmunomodulatory ingredient has anti-inflammatory activity. In someembodiments, a food, a feed, a dietary supplement, a pharmaceuticalingredient and/or a medicine provides multiple immunomodulators,including the di- or oligosaccharide having an N-acetylglucosamine unitat the reducing end (or a mixture as described herein) produced and/orpurified by a process disclosed in this specification, to adapt theimmune system for proper functioning. Immunity varies strongly indistinguishable life stages. Different food components can affectspecific immune reactions, depending on the characteristics of deviatingmetabolic processes, and consumers and patients. A food supplementedwith an immunomodulatory ingredient is also called a functional food. Afunctional food is a food product with specific health benefits forspecific groups of consumers. Examples of immunomodulatory ingredientspresent in functional food as well as in feed and dietary supplementscomprise other immunomodulatory molecules, such as di- oroligosaccharide having an N-acetylglucosamine unit at the reducing endas specified in this specification and fatty acids (PUFAs), fish oil,amino acids (such as arginine and glutamine), lectins (such asselectins), vitamins (such as vitamins A, B6, C, E, thiamine, folate)and minerals (such as zinc). Such di- or oligosaccharides having anN-acetylglucosamine unit at the reducing end may include LNB, LacNAc,poly-LacNAc and Lewis X, Lewis Y and sialyl Lewis X epitopes. Inaddition, examples of immunomodulatory ingredients present inpharmaceutical ingredients and medicines comprise other immunomodulatorymolecules, such as di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end as specified in this specification andinterleukins, lipopolysaccharides, glucan, interferon gamma and specificantibodies. Such di- or oligosaccharides having an N-acetylglucosamineunit at the reducing end present in pharmaceutical mixtures and/ormedicines may include LNB, LacNAc, poly-LacNAc and Lewis X, Lewis Y andsialyl Lewis X epitopes.

Each embodiment disclosed in the context of one aspect of thedisclosure, is also disclosed in the context of all other aspects of thedisclosure, unless explicitly stated otherwise.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described above and below are thosewell-known and commonly employed in the art. Standard techniques areused for nucleic acid and peptide synthesis. Generally, purificationsteps are performed according to the manufacturer's specifications.

Further advantages follow from the specific embodiments, the examplesand the attached drawings. It goes without saying that theabovementioned features and the features which are still to be explainedbelow can be used not only in the respectively specified combinations,but also in other combinations or on their own, without departing fromthe scope of the disclosure.

The disclosure relates to the following preferred embodiments:

1. Method for the production of a di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end, by a cell, the methodcomprising the steps of:

-   -   a. providing a cell which is capable: (i) to synthesize a        nucleotide-sugar, (ii) to synthesize N-acetylglucosamine,        and (iii) of glycosylating the N-acetylglucosamine        monosaccharide,    -   b. cultivating the cell under conditions permissive for        producing the di- or oligosaccharide,    -   c. separating the desired di- or oligosaccharide from the        cultivation.

2. The method according to embodiment 1 wherein the cell expresses atleast one glucosamine 6-phosphate N-acetyltransferase and a phosphataseto synthesize the monosaccharide N-acetylglucosamine.

3. The method according to any one of embodiments 1 and 2 wherein thecell expresses at least one glycosyltransferase to glycosylateN-acetylglucosamine.

4. The method according to any one of embodiments 1 to 3 wherein thecell is genetically modified to produce the di- or oligosaccharide.

5. The method according to any one of embodiments 1 to 4 wherein thecell is modified in the expression or activity of an enzyme selectedfrom the group comprising: glucosamine 6-phosphate N-acetyltransferase,phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.

6. The method according to any one of embodiments 1 to 5 wherein thenucleotide-sugar is selected from the group comprising: UDP-galactose(UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc),UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine(UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose(UDP-Glc), CMP-N-acetylneuraminic acid (CMP-Neu5Ac),CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate,UDP-galacturonate, GDP-rhamnose, and UDP-xylose.

7. The method according to any one of embodiments 1 to 6 wherein thenucleotide-sugar is UDP-galactose and the glycosyltransferase is anN-acetylglucosamine b-1,3-galactosyltransferase or anN-acetylglucosamine b-1,4-galactosyltransferase.

8. The method according to any one of embodiments 1 to 7 wherein thedisaccharide is lacto-N-biose (Gal-b1,3-GlcNAc) or N-acetyllactosamine(Gal-b1,4-GlcNAc), or, wherein the oligosaccharide has a lacto-N-biose(Gal-b1,3-GlcNAc) or an N-acetyllactosamine (Gal-b1,4-GlcNAc) at thereducing end.

9. The method according to any one of embodiments 1 to 8 wherein theN-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferasehaving

-   -   a. PFAM domain PF00535, and        -   i) comprises the sequence [AGPS]XXLN(X_(n))RXDXD with SEQ ID            NO:01, wherein X is any amino acid and wherein n is 12 to            17, or        -   ii) comprises the sequence            PXXLN(X_(n))RXDXD(X_(m))[FWY]XX[HKR]XX[NQST] with SEQ ID            NO:02, wherein X is any amino acid and wherein n is 12 to 17            and m 100 to 115, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NO: 03, 04, 05, 06, 07 or 08, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 03, 04, 05, 06, 07 or 08 having at least            80% overall sequence identity to the full-length of any one            of the N-acetylglucosamine b-1,3-galactosyltransferase            polypeptide with SEQ ID NO: 03, 04, 05, 06, 07 or 08 and            having N-acetylglucosamine b-1,3-galactosyltransferase            activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 03, 04, 05, 06, 07            or 08 and having N-acetylglucosamine            b-1,3-galactosyltransferase activity, or    -   b. PFAM domain IPR002659, and        -   i) comprises the sequence            KT(X_(n))[FY]XXKXDXD(X_(m))[FHY]XXG(X, no A, G,            S)(X_(p))X(no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO:09,            wherein X is any amino acid and wherein n is 13 to 16, m 35            to 70 and p 20 to 45, or        -   ii) comprises a polypeptide sequence according to any one of            SEQ ID NO: 10, 11, 12 or 13, or        -   iii) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 10, 11, 12 or 13 having at least 80%            overall sequence identity to the full-length of any one of            the N-acetylglucosamine b-1,3-galactosyltransferase            polypeptide with SEQ ID NO: 10, 11, 12 or 13 and having            N-acetylglucosamine b-1,3-galactosyltransferase activity, or        -   iv) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 10, 11, 12 or 13            and having N-acetylglucosamine b-1,3-galactosyltransferase            activity.

10. The method according to any one of embodiments 1 to 8 wherein theN-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferasehaving

-   -   a. PFAM domain PF01755, and        -   i) comprises the sequence            EXXCXXSHXX[ILV][FWY](X_(n))EDD(X_(m))[ACGST]XXYX[ILMV] with            SEQ ID NO:14, wherein X is any amino acid and wherein n is            13 to 15 and m 50 to 76, or        -   ii) comprises a polypeptide sequence according to any one of            SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, or        -   iii) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23            having at least 80% overall sequence identity to the            full-length of any one of the N-acetylglucosamine            b-1,4-galactosyltransferase polypeptide with SEQ ID NO: 15,            16, 17, 18, 19, 20, 21, 22 or 23 and having            N-acetylglucosamine b-1,4-galactosyltransferase activity, or        -   iv) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 15, 16, 17, 18, 19,            20, 21, 22 or 23 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   b. PFAM domain PF00535, and        -   i) comprises the sequence            R[KN]XXXXXXXGXXXX[FL]XDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE]            with SEQ ID NO:24, wherein X is any amino acid and wherein n            is 50 to 75 and m 10 to 30, or        -   ii) comprises the sequence            R[KN]XXXXXXXGXXXXFXDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE](X_(p))            [FWY]XX[HKR]XX[NQST] with SEQ ID NO:25, wherein X is any            amino acid and wherein n is 50 to 75, m is 10 to 30 and p is            20 to 25, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NO: 26, 27 or 28, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 26, 27 or 28 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 26, 27 or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 26, 27 or 28 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or    -   c. PFAM domain PF02709 and not having PFAM domain PF00535, and        -   i) comprises the sequence            [FWY]XX[FY][FWY](X23)[FWY][GQ]X[DE]D with SEQ ID NO:29,            wherein X is any amino acid, or        -   ii) comprises the sequence [PV]W[GHNP](X_(n))[FWY][GQ]X[DE]D            with SEQ ID NO:30, wherein X is any amino acid and wherein n            is 21 to 24, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NOs: 31, 32, 33, 34, 35 or 36, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 31, 32, 33, 34, 35 or 36 having at least            80% overall sequence identity to the full-length of any one            of the N-acetylglucosamine b-1,4-galactosyltransferase            polypeptide with SEQ ID NO: 31, 32, 33, 34, 35 or 36 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 31, 32, 33, 34, 35            or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   d. PFAM domain PF03808, and        -   i) comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA with SEQ ID            NO:37, wherein X is any amino acid and wherein n is 20 to            25, or        -   ii) comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA(X_(m))[HR]XG[FWY](X_(p))GXGXXXQ[DE]            with SEQ ID NO:38, wherein X is any amino acid and wherein n            is 20 to 25, m is 40 to 50 and p is 22 to 30, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NO: 39, 40 or 41, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 39, 40 or 41 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 39, 40 or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 39, 40 or 41 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity.

11. The method according to any one of the preceding embodiments,wherein

-   -   a. the glucosamine 6-phosphate N-acetyltransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P43577 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P43577 having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P43577 and having glucosamine 6-phosphate        N-acetyltransferase activity, and    -   b. the L-glutamine-D-fructose-6-phosphate aminotransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P17169 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P17169 having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P17169 and having        L-glutamine-D-fructose-6-phosphate aminotransferase activity; or        is a modified version that differs from the polypeptide with        UniProt ID P17169 by an A39T, an R250C and an G4725 mutation.

12. The method according to any one of the preceding embodiments,wherein the cell is capable to catabolize a carbon source selected fromthe list comprising: glucose, fructose, galactose, lactose, sucrose,maltose, malto-oligosaccharides, maltotriose, sorbitol, xylose,rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch,cellulose, hemi-cellulose, corn-steep liquor, high-fructose syrup,glycerol, acetate, citrate, lactate, pyruvate.

13. The method according to any one of the preceding embodiments,wherein the cell is unable to convert N-acetylglucosamine-6-phosphate toglucosamine-6-phosphate, and/or unable to convertglucosamine-6-phosphate to fructose-6-phoshate.

14. The method according to any one of the preceding embodiments,wherein the cell is modified to produce GDP-fucose.

15. The method according to any one of the preceding embodiments,wherein the cell is modified for enhanced GDP-fucose production andwherein the modification is chosen from the group comprising: knock-outof an UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferaseencoding gene, over-expression of a GDP-L-fucose synthase encoding gene,over-expression of a GDP-mannose 4,6-dehydratase encoding gene,over-expression of a mannose-1-phosphate guanylyltransferase encodinggene, over-expression of a phosphomannomutase encoding gene orover-expression of a mannose-6-phosphate isomerase. encoding gene.

16. The method according to any one of the preceding embodiments,wherein the cell is modified to produce UDP-galactose.

17. The method according to any one of the preceding embodiments,wherein the cell is modified for enhanced UDP-galactose production andwherein the modification is chosen from the group comprising: knock-outof an 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out ofa galactose-1-phosphate uridylyltransferase encoding gene.

18. The method according to any one of the preceding embodiments,wherein the cell is modified to produce CMP-N-acetylneuraminic acid.

19. The method according to any one of the preceding embodiments,wherein the cell is modified for enhanced CMP-N-acetylneuraminic acidproduction and wherein the modification is chosen from the groupcomprising: over-expression of an CMP-sialic acid synthetase encodinggene, over-expression of a sialate synthase encoding gene,over-expression of an N-acetyl-D-glucosamine 2-epimerase encoding gene.

20. The method according to any one of the preceding embodiments,wherein the cell is capable to express at least one furtherglycosyltransferase and wherein the further glycosyltransferase ischosen from the group comprising: fucosyltransferases,sialyltransferases, galactosyltransferases, glucosyltransferases,mannosyltransferases, N-acetylglucosaminyltransferases,N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases,xylosyltransferases, glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases.

21. The method according to embodiment 20, wherein the cell is modifiedin the expression or activity of the further glycosyltransferase.

22. The method according to any one of the preceding embodiments,wherein the oligosaccharide is chosen from the list comprising:2-fucosyl lacto-N-biose, 4-fucosyl lacto-N-biose, 2-4-difucosyllacto-N-biose, 3′-sialyl lacto-N-biose, 6′-sialyl lacto-N-biose,3′,6′-disialyl lacto-N-biose, 6,6′-disialyl lacto-N-biose,2′-fucosyl-3′-sialyl lacto-N-biose, 2′-fucosyl-6′-sialyl lacto-N-biose,4-fucosyl-3′-sialyl lacto-N-biose, 4-fucosyl-6′-sialyl lacto-N-biose,2-fucosyl N-acetyllactosamine, 3′-fucosyl N-acetyllactosamine,2,3′-difucosyl N-acetyllactosamine, 3′-sialyl N-acetyllactosamine,6′-sialyl N-acetyllactosamine, 3′,6′-disialyl N-acetyllactosamine,6,6′-disialyl N-acetyllactosamine, 2′-fucosyl-3 ‘-sialylN-acetyllactosamine, 2’-fucosyl-6′-sialyl N-acetyllactosamine,3-fucosyl-3′-sialyl N-acetyllactosamine, 3′-fucosyl-6′-sialylN-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), thexenotransplantation epitope (Gal-a1,3-Gal-b1,4-GlcNAc),Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine,GalNAc-b1,3-Gal-b1,4-GlcNAc.

23. Metabolically engineered cell capable: (i) to synthesize anucleotide-sugar, (ii) to synthesize N-acetylglucosamine, and (iii) ofglycosylating the N-acetylglucosamine monosaccharide; wherein the cellproduces a di- or oligosaccharide having an N-acetylglucosamine unit atthe reducing end.

24. Cell according to embodiment 23, wherein the cell expresses at leastone glucosamine 6-phosphate N-acetyltransferase and a phosphatase tosynthesize N-acetylglucosamine.

25. Cell according to any one of embodiments 23 and 24, wherein the cellexpresses at least one glycosyltransferase to glycosylateN-acetylglucosamine.

26. Cell according to any one of embodiments 23 to 25, wherein the cellis modified in the expression or activity of an enzyme selected from thegroup comprising: glucosamine 6-phosphate N-acetyltransferase,phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.

27. Cell according to any one of embodiments 23 to 26, wherein thenucleotide-sugar is selected from the group comprising: UDP-galactose(UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc),UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine(UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose(UDP-Glc), CMP-N-acetylneuraminic acid (CMP-Neu5Ac),CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate,UDP-galacturonate, GDP-rhamnose, and UDP-xylose.

28. Cell according to any one of embodiments 23 to 27, wherein thenucleotide-sugar is UDP-galactose and the glycosyltransferase is anN-acetylglucosamine b-1,3-galactosyltransferase or anN-acetylglucosamine b-1,4-galactosyltransferase.

29. Cell according to any one of embodiments 23 to 28, wherein thedisaccharide is lacto-N-biose (Gal-b1,3-GlcNAc) or N-acetyllactosamine(Gal-b1,4-GlcNAc), or, wherein the oligosaccharide has a lacto-N-biose(Gal-b1,3-GlcNAc) or an N-acetyllactosamine (Gal-b1,4-GlcNAc) at thereducing end.

30. Cell according to any one of embodiments 23 to 29, wherein theN-acetylglucosamine b-1,3-galactosyltransferase is a glycosyltransferasehaving

-   -   a. PFAM domain PF00535, and        -   i) comprises the sequence [AGPS]XXLN(X_(n))RXDXD with SEQ ID            NO:01, wherein X is any amino acid and wherein n is 12 to            17, or        -   ii) comprises the sequence            PXXLN(X_(n))RXDXD(X_(m))[FWY]XX[HKR]XX[NQST] with SEQ ID            NO:02, wherein X is any amino acid and wherein n is 12 to 17            and m 100 to 115, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NO: 03, 04, 05, 06, 07 or 08, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 03, 04, 05, 06, 07 or 08 having at least            80% overall sequence identity to the full-length of any one            of the N-acetylglucosamine b-1,3-galactosyltransferase            polypeptide with SEQ ID NO: 03, 04, 05, 06, 07 or 08 and            having N-acetylglucosamine b-1,3-galactosyltransferase            activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 03, 04, 05, 06, 07            or 08 and having N-acetylglucosamine            b-1,3-galactosyltransferase activity, or    -   b. PFAM domain IPR002659, and        -   i) comprises the sequence            KT(X_(n))[FY]XXKXDXD(X_(m))[FHY]XXG(X, no A, G,            S)(X_(p))X(no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO:09,            wherein X is any amino acid and wherein n is 13 to 16, m 35            to 70 and p 20 to 45, or        -   ii) comprises a polypeptide sequence according to any one of            SEQ ID NO:10, 11, 12 or 13, or        -   iii) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 10, 11, 12 or 13 having at least 80%            overall sequence identity to the full-length of any one of            the N-acetylglucosamine b-1,3-galactosyltransferase            polypeptide with SEQ ID NO: 10, 11, 12 or 13 and having            N-acetylglucosamine b-1,3-galactosyltransferase activity, or        -   iv) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 10, 11, 12 or 13            and having N-acetylglucosamine b-1,3-galactosyltransferase            activity.

31. Cell according to any one of embodiments 23 to 29, wherein theN-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferasehaving

-   -   a. PFAM domain PF01755, and        -   i) comprises the sequence            EXXCXXSHXX[ILV][FWY](X_(n))EDD(X_(m))[ACGST]XXYX[ILMV] with            SEQ ID NO:14, wherein X is any amino acid and wherein n is            13 to 15 and m 50 to 76, or        -   ii) comprises a polypeptide sequence according to any one of            SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22 or 23, or        -   iii) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23            having at least 80% overall sequence identity to the            full-length of any one of the N-acetylglucosamine            b-1,4-galactosyltransferase polypeptide with SEQ ID NO: 15,            16, 17, 18, 19, 20, 21, 22 or 23 and having            N-acetylglucosamine b-1,4-galactosyltransferase activity, or        -   iv) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 15, 16, 17, 18, 19,            20, 21, 22 or 23 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   b. PFAM domain PF00535, and        -   i) comprises the sequence            R[KN]XXXXXXXGXXXX[FL]XDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE]            with SEQ ID NO:24, wherein X is any amino acid and wherein n            is 50 to 75 and m 10 to 30, or        -   ii) comprises the sequence            R[KN]XXXXXXXGXXXXFXDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE](X_(p))            [FWY]XX[HKR]XX[NQST] with SEQ ID NO:25, wherein X is any            amino acid and wherein n is 50 to 75, m is 10 to 30 and p is            20 to 25, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NO:26, 27 or 28, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 26, 27 or 28 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 26, 27 or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 26, 27 or 28 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or    -   c. PFAM domain PF02709 and not having PFAM domain PF00535,        -   i) comprises the sequence            [FWY]XX[FY][FWY](X23)[FWY][GQ]X[DE]D with SEQ ID NO:29,            wherein X is any amino acid, or        -   ii) comprises the sequence [PV]W[GHNP](X_(n))[FWY][GQ]X[DE]D            with SEQ ID NO:30, wherein X is any amino acid and wherein n            is 21 to 24, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NO:31, 32, 33, 34, 35 or 36, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 31, 32, 33, 34, 35 or 36 having at least            80% overall sequence identity to the full-length of any one            of the N-acetylglucosamine b-1,4-galactosyltransferase            polypeptide with SEQ ID NO: 31, 32, 33, 34, 35 or 36 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 31, 32, 33, 34, 35            or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   d. PFAM domain PF03808, and        -   i) comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA with SEQ ID            NO:37, wherein X is any amino acid and wherein n is 20 to            25, or        -   ii) comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA(X_(m))[HR]XG[FWY](Xp)GXGXXXQ[DE]            with SEQ ID NO:38, wherein X is any amino acid and wherein n            is 20 to 25, m is 40 to 50 and p is 22 to 30, or        -   iii) comprises a polypeptide sequence according to any one            of SEQ ID NO: 39, 40 or 41, or        -   iv) is a functional homologue, variant or derivative of any            one of SEQ ID NO: 39, 40 or 41 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 39, 40 or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v) comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 39, 40 or 41 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity.

32. The cell according to any one of embodiments 23 to 31, wherein

-   -   a. the glucosamine 6-phosphate N-acetyltransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P43577 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P43577 having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P43577 and having glucosamine 6-phosphate        N-acetyltransferase activity, and    -   b. the L-glutamine-D-fructose-6-phosphate aminotransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P17169 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P17169 having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P17169 and having        L-glutamine-D-fructose-6-phosphate aminotransferase activity; or        is a modified version that differs from the polypeptide with        UniProt ID P17169by an A39T, an R250C and an G472S mutation.

33. The cell according to any one of embodiments 23 to 32, wherein thecell is capable to catabolize a carbon source selected from the listcomprising: glucose, fructose, galactose, lactose, sucrose, maltose,malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose,mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose,hemi-cellulose, corn-steep liquor, high-fructose syrup, glycerol,acetate, citrate, lactate, pyruvate.

34. The cell according to any one of embodiments 23 to 33, wherein thecell is unable to convert N-acetylglucosamine-6-phosphate toglucosamine-6-phosphate, and/or unable to convertglucosamine-6-phosphate to fructose-6-phoshate.

35. The cell according to any one of embodiments 23 to 34, wherein thecell is modified to produce GDP-fucose.

36. The cell according to any one of embodiments 23 to 35, wherein thecell is modified for enhanced GDP-fucose production and wherein themodification is chosen from the group comprising: knock-out of anUDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferaseencoding gene, over-expression of a GDP-L-fucose synthase encoding gene,over-expression of a GDP-mannose 4,6-dehydratase encoding gene,over-expression of a mannose-1-phosphate guanylyltransferase encodinggene, over-expression of a phosphomannomutase encoding gene orover-expression of a mannose-6-phosphate isomerase-encoding gene.

37. The cell according to any one of embodiments 23 to 36, wherein thecell is modified to produce UDP-galactose.

38. The cell according to any one of embodiments 23 to 37, wherein thecell is modified for enhanced UDP-galactose production and wherein themodification is chosen from the group comprising: knock-out of an5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of agalactose-1-phosphate uridylyltransferase encoding gene.

39. The cell according to any one of embodiments 23 to 38, wherein thecell is modified to produce CMP-N-acetylneuraminic acid.

40. The cell according to any one of embodiments 23 to 39, wherein thecell is modified for enhanced CMP-N-acetylneuraminic acid production andwherein the modification is chosen from the group comprising:over-expression of an CMP-sialic acid synthetase encoding gene,over-expression of a sialate synthase encoding gene, over-expression ofan N-acetyl-D-glucosamine 2-epimerase encoding gene.

41. The cell according to any one of embodiments 23 to 40, wherein thecell is capable to express least one further glycosyltransferase andwherein the further glycosyltransferase is chosen from the groupcomprising: fucosyltransferases, sialyltransferases,galactosyltransferases, glucosyltransferases, mannosyltransferases,N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases,N-acetylmannosaminyltransferases, xylosyltransferases,glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases.

42. The cell according to embodiment 41, wherein the cell is modified inthe expression or activity of the further glycosyltransferase.

43. The cell according to any one of embodiments 23 to 41, wherein theoligosaccharide is chosen from the list comprising: 2-fucosyllacto-N-biose, 4-fucosyl lacto-N-biose, 2-4-difucosyl lacto-N-biose,3′-sialyl lacto-N-biose, 6′-sialyl lacto-N-biose, 3′,6′-disialyllacto-N-biose, 6,6′-disialyl lacto-N-biose, 2′-fucosyl-3′-sialyllacto-N-biose, 2′-fucosyl-6′-sialyl lacto-N-biose, 4-fucosyl-3′-sialyllacto-N-biose, 4-fucosyl-6′-sialyl lacto-N-biose, 2-fucosylN-acetyllactosamine, 3′-fucosyl N-acetyllactosamine, 2,3′-difucosylN-acetyllactosamine, 3′-sialyl N-acetyllactosamine, 6′-sialylN-acetyllactosamine, 3′,6′-disialyl N-acetyllactosamine, 6,6′-disialylN-acetyllactosamine, 2′-fucosyl-3′-sialyl N-acetyllactosamine,2′-fucosyl-6′-sialyl N-acetyllactosamine, 3-fucosyl-3′-sialylN-acetyllactosamine, 3′-fucosyl-6′-sialyl N-acetyllactosamine, P1trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), the xenotransplantationepitope (Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc,poly-N-acetyllactosamine, GalNAc-b 1,3-Gal-b1,4-GlcNAc.

44. The cell according to any one of embodiments 23 to 43 or methodaccording to any one of embodiments 1 to 22, wherein the cell isselected from the group consisting of microorganism, plant, or animalcells, preferably the microorganism is a bacterium, fungus or a yeast,preferably the plant is a rice, cotton, rapeseed, soy, maize or cornplant, preferably the animal is an insect, fish, bird or non-humanmammal; preferably the cell is an Escherichia coli cell.

45. The cell according to any one of embodiments 23 to 44 or methodaccording to any one of embodiments 1 to 22, or 44, wherein the cell isa cell of a bacterium, preferably of an Escherichia coli strain, morepreferably of an Escherichia coli strain which is a K12 strain, evenmore preferably the Escherichia coli K12 strain is Escherichia coliMG1655.

46. The cell according to any one of embodiments 22 to 45 or methodaccording to any one of embodiments 1 to 21, 44 or 45, wherein the cellis a yeast cell.

47. The method according to any one of embodiments 1 to 22, 44 to 46,wherein the separation comprises at least one of the following steps:clarification, ultrafiltration, nanofiltration, reverse osmosis,microfiltration, activated charcoal or carbon treatment, tangential flowhigh-performance filtration, tangential flow ultrafiltration, affinitychromatography, ion exchange chromatography, hydrophobic interactionchromatography and/or gel filtration, ligand exchange chromatography.

48. The method according to any one of embodiments 1 to 22, 44 to 47,further comprising purification of the di- or oligosaccharide from thecell.

49. The method according to any one of embodiments 1 to 22, 44 to 48,wherein the purification comprises at least one of the following steps:use of activated charcoal or carbon, use of charcoal, nanofiltration,ultrafiltration or ion exchange, use of alcohols, use of aqueous alcoholmixtures, crystallization, evaporation, precipitation, drying, spraydrying or lyophilization.

50. Use of a cell according to any one of embodiments 23 to 46 for theproduction of a di- or oligosaccharide having an N-acetylglucosamineunit at the reducing end, preferably for the production of LNB orLacNAc, more preferably for the production of sialylated or fucosylatedforms of LNB or LacNAc.

Moreover, the disclosure relates to the following preferred specificembodiments:

1. Method for the production of an oligo- or disaccharide having anN-acetylglucosamine unit at the reducing end, by a cell, preferably asingle cell, the method comprising the steps of:

-   -   a. providing a cell which is capable of: (i) synthesizing a        nucleotide-sugar and the monosaccharide N-acetylglucosamine        (GlcNAc) and (ii) expressing a glycosyltransferase to        glycosylate the GlcNAc monosaccharide to produce the di- or        oligosaccharide,    -   b. cultivating the cell under conditions permissive for        producing the di- or oligosaccharide,    -   c. preferably, separating the di- or oligosaccharide from the        cultivation.

2. Method for the production of an oligosaccharide having anN-acetylglucosamine unit at the reducing end, by a cell, the methodcomprising the steps of:

-   -   a. providing a cell which is capable of: (i) synthesizing a        nucleotide-sugar and the monosaccharide N-acetylglucosamine        (GlcNAc) and (ii) expressing a glycosyltransferase to        glycosylate the GlcNAc monosaccharide to produce the        oligosaccharide,    -   b. cultivating the cell under conditions permissive for        producing the oligosaccharide,    -   c. preferably, separating the oligosaccharide from the        cultivation.

3. Method for the production of a mixture comprising (i) a di- and/oroligosaccharide having an N-acetylglucosamine unit at the reducing endand (ii) one or more lactose-based mammalian milk oligosaccharides(MMOs), by a cell, the method comprising the steps of:

-   -   a. providing a cell which is capable of: (i) synthesizing a        nucleotide-sugar and the monosaccharide N-acetylglucosamine        (GlcNAc) and (ii) expressing a glycosyltransferase to        glycosylate the GlcNAc monosaccharide to produce the di- and/or        oligosaccharide having an N-acetylglucosamine unit at the        reducing end,    -   b. cultivating the cell under conditions permissive for        producing the mixture,    -   c. preferably, separating the mixture from the cultivation.

4. The method according to any one of previous specific embodiments,wherein the cell expresses at least one glucosamine 6-phosphateN-acetyltransferase and a phosphatase to synthesize the monosaccharideN-acetylglucosamine.

5. The method according to any one of previous specific embodiments,wherein the cell expresses at least one glycosyltransferase toglycosylate N-acetylglucosamine.

6. The method according to any one of previous specific embodiments,wherein the cell is genetically modified to produce the di- oroligosaccharide.

7. The method according to specific embodiment 6, wherein the cell ismodified with one or more gene expression modules, characterized in thatthe expression from any of the expression modules is either constitutiveor is created by a natural inducer.

8. The method according to any one of specific embodiment 6 or 7,wherein the cell comprises multiple copies of the same coding DNAsequence encoding for one protein.

9. The method according to any one of previous specific embodiments,wherein the cell is modified in the expression or activity of an enzymeselected from the group comprising: glucosamine 6-phosphateN-acetyltransferase, phosphatase, glycosyltransferase,L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose4-epimerase.

10. The method according to any one of previous specific embodiments,wherein the nucleotide-sugar is selected from the group comprising:UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc),UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine(UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose(UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, and UDP-xylose.

11. The method according to any one of previous specific embodiments,wherein the nucleotide-sugar is UDP-galactose and theglycosyltransferase is an N-acetylglucosamineb-1,3-galactosyltransferase or an N-acetylglucosamineb-1,4-galactosyltransferase.

12. The method according to any one of previous specific embodiments,wherein the oligosaccharide has a lacto-N-biose (Gal-b1,3-GlcNAc) or anN-acetyllactosamine (Gal-b1,4-GlcNAc) at the reducing end.

13. The method according to any one of previous specific embodiments,wherein the N-acetylglucosamine b-1,3-galactosyltransferase is aglycosyltransferase having

-   -   a. PFAM domain PF00535, and        -   i. comprises the sequence [AGPS]XXLN(X_(n))RXDXD with SEQ ID            NO:01, wherein X is any amino acid and wherein n is 12 to            17, or        -   ii. comprises the sequence            PXXLN(X_(n))RXDXD(X_(m))[FWY]XX[HKR]XX[NQST] with SEQ ID            NO:02, wherein X is any amino acid and wherein n is 12 to 17            and m 100 to 115, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any one            of SEQ ID NO:03, 04, 05, 06 or 07, more preferably any one            of SEQ ID NO:03, 06 or 07, most preferably any one of SEQ ID            NO:03 or 06, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any            one of SEQ ID NO:03, 04, 05, 06 or 07, more preferably any            one of SEQ ID NO:03, 06 or 07, most preferably any one of            SEQ ID NO:03 or 06, having at least 80% overall sequence            identity to the full-length of any one of the            N-acetylglucosamine b-1,3-galactosyltransferase polypeptide            with SEQ ID NO: 03, 04, 05, 06, 07 or 08, preferably any one            of SEQ ID NO:03, 04, 05, 06 or 07, more preferably any one            of SEQ ID NO:03, 06 or 07, most preferably any one of SEQ ID            NO:03 or 06, and having N-acetylglucosamine            b-1,3-galactosyltransferase activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 03, 04, 05, 06, 07            or 08, preferably of any one of SEQ ID NO:03, 04, 05, 06 or            07, more preferably any one of SEQ ID NO:03, 06 or 07, most            preferably any one of SEQ ID NO:03 or 06, and having            N-acetylglucosamine b-1,3-galactosyltransferase activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 03,            04, 05, 06, 07 or 08, preferably of any one of SEQ ID NO:03,            04, 05, 06 or 07, more preferably any one of SEQ ID NO:03,            06 or 07, most preferably any one of SEQ ID NO:03 or 06, and            having N-acetylglucosamine b-1,3-galactosyltransferase            activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            03, 04, 05, 06, 07 or 08, preferably of any one of SEQ ID            NO:03, 04, 05, 06 or 07, more preferably any one of SEQ ID            NO:03, 06 or 07, most preferably any one of SEQ ID NO:03 or            06, and having N-acetylglucosamine            b-1,3-galactosyltransferase activity, or    -   b. PFAM domain IPR002659, and        -   i. comprises the sequence            KT(X_(n))[FY]XXKXDXD(X_(m))[FHY]XXG(X, no A, G,            S)(X_(p))X(no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO:09,            wherein X is any amino acid and wherein n is 13 to 16, m 35            to 70 and p 20 to 45, or        -   ii. comprises a polypeptide sequence according to any one of            SEQ ID NO: 10, 11, 12 or 13, or        -   iii. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 10, 11, 12 or 13 having at least 80%            overall sequence identity to the full-length of any one of            the N-acetylglucosamine b-1,3-galactosyltransferase            polypeptide with SEQ ID NO: 10, 11, 12 or 13 and having            N-acetylglucosamine b-1,3-galactosyltransferase activity, or        -   iv. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 10, 11, 12 or 13            and having N-acetylglucosamine b-1,3-galactosyltransferase            activity, or        -   v. is a functional fragment of any one of SEQ ID NO: 10, 11,            12 or 13 and having N-acetylglucosamine            b-1,3-galactosyltransferase activity, or        -   vi. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            10, 11, 12 or 13 and having N-acetylglucosamine            b-1,3-galactosyltransferase activity.

14. The method according to any one of previous specific embodiments 1to 12 wherein the N-acetylglucosamine b-1,4-galactosyltransferase is aglycosyltransferase having

-   -   a. PFAM domain PF01755, and        -   i. comprises the sequence            EXXCXXSHXX[ILV][FWY](X_(n))EDD(X_(m))[ACGST]XXYX[ILMV] with            SEQ ID NO:14, wherein X is any amino acid and wherein n is            13 to 15 and m 50 to 76, or        -   ii. comprises a polypeptide sequence according to any one of            SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably            any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more            preferably any one of SEQ ID NO: 17, 18, 20 or 21, or        -   iii. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23,            preferably any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21,            more preferably any one of SEQ ID NO: 17, 18, 20 or 21,            having at least 80% overall sequence identity to the            full-length of any one of the N-acetylglucosamine            b-1,4-galactosyltransferase polypeptide with SEQ ID NO: 15,            16, 17, 18, 19, 20, 21, 22 or 23, preferably any one of SEQ            ID NO: 15, 16, 17, 18, 20 or 21, more preferably any one of            SEQ ID NO: 17, 18, 20 or 21, and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   iv. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 15, 16, 17, 18, 19,            20, 21, 22 or 23, preferably of any one of SEQ ID NO: 15,            16, 17, 18, 20 or 21, more preferably of any one of SEQ ID            NO: 17, 18, 20 or 21, and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v. is a functional fragment of any one of SEQ ID NO: 15, 16,            17, 18, 19, 20, 21, 22 or 23, preferably of any one of SEQ            ID NO: 15, 16, 17, 18, 20 or 21, more preferably of any one            of SEQ ID NO: 17, 18, 20 or 21, and having            N-acetylglucosamine b-1,4-galactosyltransferase activity, or        -   vi. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably of any one            of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably of            any one of SEQ ID NO: 17, 18, 20 or 21, and having            N-acetylglucosamine b-1,4-galactosyltransferase activity, or    -   b. PFAM domain PF00535, and        -   i. comprises the sequence            R[KN]XXXXXXXGXXXX[FL]XDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE]            with SEQ ID NO:24, wherein X is any amino acid and wherein n            is 50 to 75 and m 10 to 30, or        -   ii. comprises the sequence            R[KN]XXXXXXXGXXXXFXDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE](X_(p))            [FWY]XX[HKR]XX[NQST] with SEQ ID NO:25, wherein X is any            amino acid and wherein n is 50 to 75, m is 10 to 30 and p is            20 to 25, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NO:26, 27 or 28, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 26, 27 or 28 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 26, 27 or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 26, 27 or 28 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 26, 27            or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            26, 27 or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   c. PFAM domain PF02709 and not having PFAM domain PF00535,        -   i. comprises the sequence            [FWY]XX[FY][FWY](X₂₃)[FWY][GQ]X[DE]D with SEQ ID NO:29,            wherein X is any amino acid, or        -   ii. comprises the sequence [PV]W[GHNP](X_(n))[FWY][GQ]X[DE]D            with SEQ ID NO:30, wherein X is any amino acid and wherein n            is 21 to 24, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NOs: 31, 32, 33, 34, 35 or 36, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 31, 32, 33, 34, 35 or 36 having at least            80% overall sequence identity to the full-length of any one            of the N-acetylglucosamine b-1,4-galactosyltransferase            polypeptide with SEQ ID NO: 31, 32, 33, 34, 35 or 36 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 31, 32, 33, 34, 35            or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 31,            32, 33, 34, 35 or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            31, 32, 33, 34, 35 or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   d. PFAM domain PF03808, and        -   i. comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA with SEQ ID            NO:37, wherein X is any amino acid and wherein n is 20 to            25, or        -   ii. comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA(X_(m))[HR]XG[FWY](Xp)GXGXXXQ[DE]            with SEQ ID NO:38, wherein X is any amino acid and wherein n            is 20 to 25, m is 40 to 50 and p is 22 to 30, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NO: 39, 40 or 41, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 39, 40 or 41 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 39, 40 or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 39, 40 or 41 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 39, 40            or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            39, 40 or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity.

15. The method according to any one of the previous specificembodiments, wherein

-   -   a. the glucosamine 6-phosphate N-acetyltransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P43577 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P43577 having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P43577 and having glucosamine 6-phosphate        N-acetyltransferase activity, and    -   b. the L-glutamine-D-fructose-6-phosphate aminotransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P17169 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P17169 having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P17169 and having        L-glutamine-D-fructose-6-phosphate aminotransferase activity; or        is a modified version that differs from the polypeptide with        UniProt ID P17169 by an A39T, an R250C and an G472S mutation.

16. The method according to any one of the previous specificembodiments, wherein the cell is capable to catabolize a carbon sourceselected from the list comprising: glucose, fructose, galactose,lactose, sucrose, maltose, malto-oligosaccharides, maltotriose,sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose,trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor,high-fructose syrup, molasses, glycerol, acetate, citrate, lactate,pyruvate.

17. The method according to any one of the previous specificembodiments, wherein the cell is unable to convertN-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/orunable to convert glucosamine-6-phosphate to fructose-6-phoshate.

18. The method according to any one of the previous specificembodiments, wherein the cell is modified to produce GDP-fucose.

19. The method according to any one of the previous specificembodiments, wherein the cell is modified for enhanced GDP-fucoseproduction and wherein the modification is chosen from the groupcomprising: knock-out of an UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase encoding gene, over-expression of aGDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose4,6-dehydratase encoding gene, over-expression of a mannose-1-phosphateguanylyltransferase encoding gene, over-expression of aphosphomannomutase encoding gene or over-expression of amannose-6-phosphate isomerase-encoding gene.

20. The method according to any one of the previous specificembodiments, wherein the cell is modified to produce UDP-galactose.

21. The method according to any one of the previous specificembodiments, wherein the cell is modified for enhanced UDP-galactoseproduction and wherein the modification is chosen from the groupcomprising: knock-out of an 5′-nucleotidase/UDP-sugar hydrolase encodinggene or knock-out of a galactose-1-phosphate uridylyltransferaseencoding gene.

22. The method according to any one of the previous specificembodiments, wherein the cell is modified to produceCMP-N-acetylneuraminic acid.

23. The method according to any one of the previous specificembodiments, wherein the cell is modified for enhancedCMP-N-acetylneuraminic acid production and wherein the modification ischosen from the group comprising: over-expression of an CMP-sialic acidsynthetase encoding gene, over-expression of a sialate synthase encodinggene, over-expression of an N-acetyl-D-glucosamine 2-epimerase encodinggene.

24. The method according to any one of the previous specificembodiments, wherein the cell is capable to express at least one furtherglycosyltransferase and wherein the further glycosyltransferase ischosen from the group comprising: fucosyltransferases,sialyltransferases, galactosyltransferases, glucosyltransferases,mannosyltransferases, N-acetylglucosaminyltransferases,N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases,xylosyltransferases, glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases, N-acetylrhamnosyltransferases,UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases,UDP-N-acetylglucosamine enolpyruvyl transferases andfucosaminyltransferases,

-   -   preferably, the fucosyltransferase is chosen from the list        comprising alpha-1,2-fucosyltransferase,        alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and        alpha-1,6-fucosyltransferase,    -   preferably, the sialyltransferase is chosen from the list        comprising alpha-2,3-sialyltransferase,        alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase,    -   preferably, the galactosyltransferase is chosen from the list        comprising beta-1,3-galactosyltransferase, N-acetylglucosamine        beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase,        N-acetylglucosamine beta-1,4-galactosyltransferase,        alpha-1,3-galactosyltransferase and        alpha-1,4-galactosyltransferase,    -   preferably, the glucosyltransferase is chosen from the list        comprising alpha-glucosyltransferase,        beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and        beta-1,4-glucosyltransferase,    -   preferably, the mannosyltransferase is chosen from the list        comprising alpha-1,2-mannosyltransferase,        alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase,    -   preferably, the N-acetylglucosaminyltransferase is chosen from        the list comprising galactoside        beta-1,3-N-acetylglucosaminyltransferase and        beta-1,6-N-acetylglucosaminyltransferase,    -   preferably, the N-acetylgalactosaminyltransferase is an        alpha-1,3-N-acetylgalactosaminyltransferase.

25. The method according to specific embodiment 24, wherein the cell ismodified in the expression or activity of the furtherglycosyltransferase.

26. The method according to any one of previous specific embodiments,wherein the cell is using one or more precursor(s) for the production ofthe di- or oligosaccharide having a GlcNAc unit at the reducing end, theprecursor(s) being fed to the cell from the cultivation medium.

27. The method according to any one of previous specific embodiments,wherein the cell is producing one or more precursor(s) for theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

28. The method according to any one of specific embodiments 26 or 27,wherein the precursor for the production of the di- or oligosaccharideis completely converted into the di- or oligosaccharide having a GlcNAcunit at the reducing end.

29. The method according to any one of previous specific embodiments,wherein the cell produces the di- or oligosaccharide having a GlcNAcunit at the reducing end intracellularly and wherein a fraction orsubstantially all of the produced di- or oligosaccharide having a GlcNAcunit at the reducing end remains intracellularly and/or is excretedoutside the cell via passive or active transport.

30. The method according to any one of previous specific embodiments,wherein the cell expresses a membrane transporter protein or apolypeptide having transport activity hereby transporting compoundsacross the outer membrane of the cell wall,

-   -   preferably, the cell is modified in the expression or activity        of the membrane transporter protein or polypeptide having        transport activity.

31. The method according to specific embodiment 30, wherein the membranetransporter protein or polypeptide having transport activity is chosenfrom the list comprising porters, P—P-bond-hydrolysis-driventransporters, β-barrel porins, auxiliary transport proteins, putativetransport proteins and phosphotransfer-driven group translocators,

-   -   preferably, the porters comprise MFS transporters, sugar efflux        transporters and siderophore exporters,    -   preferably, the P—P-bond-hydrolysis-driven transporters comprise        ABC transporters and siderophore exporters.

32. The method according to any one of specific embodiments 30 or 31,wherein the membrane transporter protein or polypeptide having transportactivity controls the flow over the outer membrane of the cell wall ofthe di- or oligosaccharide having a GlcNAc unit at the reducing endand/or of one or more precursor(s) and/or acceptor(s) to be used in theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

33. The method according to any one of specific embodiments 30 to 32,wherein the membrane transporter protein or polypeptide having transportactivity provides improved production and/or enabled and/or enhancedefflux of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

34. The method according to any one of the specific embodiments 6 to 33,wherein the cell comprises a modification for reduced production ofacetate compared to a non-modified progenitor.

35. The method according to specific embodiment 34, wherein the cellcomprises a lower or reduced expression and/or abolished, impaired,reduced or delayed activity of any one or more of the proteinscomprising beta-galactosidase, galactoside O-acetyltransferase,N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphatedeaminase, N-acetylglucosamine repressor, ribonucleotidemonophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase,N-acetylneuraminate lyase, N-acetylmannosamine kinase,N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man,EIID-Man, ushA, galactose-1-phosphate uridylyltransferase,glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase,ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobicrespiration control protein, transcriptional repressor IclR, lonprotease, glucose-specific translocating phosphotransferase enzyme IIBCcomponent ptsG, glucose-specific translocating phosphotransferase (PTS)enzyme IIBC component malX, enzyme IIA^(Glc), beta-glucoside specificPTS enzyme II, fructose-specific PTS multiphosphoryl transfer proteinFruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase,pyruvate-formate lyase, acetate kinase, phosphoacyltransferase,phosphate acetyltransferase, pyruvate decarboxylase compared to anon-modified progenitor.

36. The method according to any one of the previous specificembodiments, wherein the cell is capable to produce phosphoenolpyruvate(PEP).

37. The method according to any one of the specific embodiments 6 to 36,wherein the cell is modified for enhanced production and/or supply ofphosphoenolpyruvate (PEP) compared to a non-modified progenitor.

38. The method according to any one of the specific embodiments 6 to 37,wherein the cell comprises a catabolic pathway for selected mono-, di-or oligosaccharides which is at least partially inactivated, the mono-,di-, or oligosaccharides being involved in and/or required for theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

39. The method according to any one of the previous specificembodiments, wherein the cell resists the phenomenon of lactose killingwhen grown in an environment in which lactose is combined with one ormore other carbon source(s).

40. The method according to any one of the previous specificembodiments, wherein the cell produces 90 g/L or more of the di- oroligosaccharide having a GlcNAc at the reducing end in the whole brothand/or supernatant and/or wherein the di- or oligosaccharide having aGlcNAc at the reducing end in the whole broth and/or supernatant has apurity of at least 80% measured on the total amount of the di- oroligosaccharide having a GlcNAc unit at the reducing end and itsprecursor(s) in the whole broth and/or supernatant, respectively.

41. The method according to any one of the previous specificembodiments, wherein the cell is stably cultured in a medium.

42. Method according to any one of the previous specific embodiments,wherein the conditions comprise:

-   -   use of a culture medium comprising at least one precursor and/or        acceptor for the production of the di- or oligosaccharide having        a GlcNAc unit at the reducing end, and/or    -   adding to the culture medium at least one precursor and/or        acceptor feed for the production of the di- or oligosaccharide        having a GlcNAc unit at the reducing end.

43. Method according to any one of the previous specific embodiments,the method comprising at least one of the following steps:

-   -   i) Use of a culture medium comprising at least one precursor        and/or acceptor;    -   ii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed wherein the total reactor volume        ranges from 250 mL (milliliter) to 10.000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        two-fold of the volume of the culture medium before the addition        of the precursor and/or acceptor feed;    -   iii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed wherein the total reactor volume        ranges from 250 mL (milliliter) to 10.000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        two-fold of the volume of the culture medium before the addition        of the precursor and/or acceptor feed and wherein preferably,        the pH of the precursor and/or acceptor feed is set between 3        and 7 and wherein preferably, the temperature of the precursor        and/or acceptor feed is kept between 20° C. and 80° C.;    -   iv) Adding at least one precursor and/or acceptor feed in a        continuous manner to the culture medium over the course of 1        day, 2 days, 3 days, 4 days, 5 days by means of a feeding        solution;    -   v) Adding at least one precursor and/or acceptor feed in a        continuous manner to the culture medium over the course of 1        day, 2 days, 3 days, 4 days, 5 days by means of a feeding        solution and wherein preferably, the pH of the feeding solution        is set between 3 and 7 and wherein preferably, the temperature        of the feeding solution is kept between 20° C. and 80° C.;

the method resulting in the di- or oligosaccharide having a GlcNAc unitat the reducing end with a concentration of at least 50 g/L, preferablyat least 75 g/L, more preferably at least 90 g/L, more preferably atleast 100 g/L, more preferably at least 125 g/L, more preferably atleast 150 g/L, more preferably at least 175 g/L, more preferably atleast 200 g/L in the final cultivation.

44. Method according to any one of the specific embodiments 1 to 42, themethod comprising at least one of the following steps:

-   -   i) Use of a culture medium comprising at least 50, more        preferably at least 75, more preferably at least 100, more        preferably at least 120, more preferably at least 150 gram of        lactose per liter of initial reactor volume wherein the reactor        volume ranges from 250 mL to 10.000 m³ (cubic meter);    -   ii) Adding to the culture medium a lactose feed comprising at        least 50, more preferably at least 75, more preferably at least        100, more preferably at least 120, more preferably at least 150        gram of lactose per liter of initial reactor volume wherein the        reactor volume ranges from 250 mL to 10.000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        2-fold of the volume of the culture medium before the addition        of the lactose feed;    -   iii) Adding to the culture medium a lactose feed comprising at        least 50, more preferably at least 75, more preferably at least        100, more preferably at least 120, more preferably at least 150        gram of lactose per liter of initial reactor volume wherein the        reactor volume ranges from 250 mL to 10.000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        2-fold of the volume of the culture medium before the addition        of the lactose feed and wherein preferably the pH of the lactose        feed is set between 3 and 7 and wherein preferably the        temperature of the lactose feed is kept between 20° C. and 80°        C.;    -   iv) Adding a lactose feed in a continuous manner to the culture        medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days        by means of a feeding solution;    -   v) Adding a lactose feed in a continuous manner to the culture        medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days        by means of a feeding solution and wherein the concentration of        the lactose feeding solution is 50 g/L, preferably 75 g/L, more        preferably 100 g/L, more preferably 125 g/L, more preferably 150        g/L, more preferably 175 g/L, more preferably 200 g/L, more        preferably 225 g/L, more preferably 250 g/L, more preferably 275        g/L, more preferably 300 g/L, more preferably 325 g/L, more        preferably 350 g/L, more preferably 375 g/L, more preferably,        400 g/L, more preferably 450 g/L, more preferably 500 g/L, even        more preferably, 550 g/L, most preferably 600 g/L; and wherein        preferably the pH of the feeding solution is set between 3 and 7        and wherein preferably the temperature of the feeding solution        is kept between 20° C. and 80° C.;

the method resulting in the di- or oligosaccharide having a GlcNAc unitat the reducing end with a concentration of at least 50 g/L, preferablyat least 75 g/L, more preferably at least 90 g/L, more preferably atleast 100 g/L, more preferably at least 125 g/L, more preferably atleast 150 g/L, more preferably at least 175 g/L, more preferably atleast 200 g/L in the final volume of the cultivation.

45. Method according to specific embodiment 44, wherein the lactose feedis accomplished by adding lactose from the beginning of the cultivationin a concentration of at least 5 mM, preferably in a concentration of30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in aconcentration >300 mM.

46. Method according to any one of specific embodiment 44 or 45, whereinthe lactose feed is accomplished by adding lactose to the cultivation ina concentration, such, that throughout the production phase of thecultivation a lactose concentration of at least 5 mM, preferably 10 mMor 30 mM is obtained.

47. Method according to any one of the previous specific embodiments,wherein the cells are cultivated for at least about 60, 80, 100, orabout 120 hours or in a continuous manner.

48. Method according to any one of the previous specific embodiments,wherein the culture medium contains at least one precursor selected fromthe group comprising lactose, galactose, fucose and sialic acid.

49. Method according to any one of the previous specific embodiments,wherein a first phase of exponential cell growth is provided by adding acarbon-based substrate, preferably glucose or sucrose, to the culturemedium comprising a precursor, followed by a second phase wherein only acarbon-based substrate, preferably glucose or sucrose, is added to theculture medium.

50. Method according to any one of the specific embodiments 1 to 49,wherein a first phase of exponential cell growth is provided by adding acarbon-based substrate, preferably glucose or sucrose, to the culturemedium comprising a precursor, followed by a second phase wherein acarbon-based substrate, preferably glucose or sucrose, and a precursorare added to the culture medium.

51. The method according to any one of the previous specificembodiments, wherein the cell produces a mixture of charged, preferablysialylated, and/or neutral di- and oligosaccharides comprising at leastone di- or oligosaccharide having a GlcNAc unit at the reducing end.

52. The method according to any one of the previous specificembodiments, wherein the cell produces a mixture of charged, preferablysialylated, and/or neutral oligosaccharides comprising at leastoligosaccharide having a GlcNAc unit at the reducing end.

53. The method according to any one of the previous specificembodiments, wherein the oligosaccharide is chosen from the listcomprising: 2-fucosyl lacto-N-biose, 4-fucosyl lacto-N-biose,2-4-difucosyl lacto-N-biose, 3′-sialyl lacto-N-biose, 6′-sialyllacto-N-biose, 3′,6′-disialyl lacto-N-biose, 6,6′-disialyllacto-N-biose, 2′-fucosyl-3′-sialyl lacto-N-biose, 2′-fucosyl-6′-sialyllacto-N-biose, 4-fucosyl-3′-sialyl lacto-N-biose, 4-fucosyl-6′-sialyllacto-N-biose, 2-fucosyl N-acetyllactosamine, 3′-fucosylN-acetyllactosamine, 2,3′-difucosyl N-acetyllactosamine, 3′-sialylN-acetyllactosamine, 6′-sialyl N-acetyllactosamine, 3′,6′-disialylN-acetyllactosamine, 6,6′-disialyl N-acetyllactosamine,2′-fucosyl-3′-sialyl N-acetyllactosamine, 2′-fucosyl-6′-sialylN-acetyllactosamine, 3-fucosyl-3′-sialyl N-acetyllactosamine,3′-fucosyl-6′-sialyl N-acetyllactosamine, P1 trisaccharide(Gal-a1,4-Gal-b1,4-GlcNAc), the xenotransplantation epitope(Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc,poly-N-acetyllactosamine, GalNAc-b1,3-Gal-b1,4-GlcNAc.

54. The method according to any one of the specific embodiments 1 or 3to 53, wherein the disaccharide having a GlcNAc unit at the reducing enddoes not comprise chitobiose (GlcNAc-GlcNAc).

55. The method according to any one of the previous specificembodiments, wherein the oligosaccharide having a GlcNAc unit at thereducing end does not comprise a chitobiose at the reducing end,preferably does not comprise an N-glycan.

56. Metabolically engineered cell for the production of an oligo- ordisaccharide having an N-acetylglucosamine unit at the reducing end,wherein the cell is capable of: (i) synthesizing a nucleotide-sugar andthe monosaccharide N-acetylglucosamine (GlcNAc) and (ii) expressing aglycosyltransferase to glycosylate the GlcNAc monosaccharide to producethe di- or oligosaccharide.

57. Metabolically engineered cell for the production of anoligosaccharide having an N-acetylglucosamine unit at the reducing end,wherein the cell is capable of: (i) synthesizing a nucleotide-sugar andthe monosaccharide N-acetylglucosamine (GlcNAc) and (ii) expressing aglycosyltransferase to glycosylate the GlcNAc monosaccharide to producethe oligosaccharide.

58. Metabolically engineered cell for the production of a mixturecomprising (i) a di- and/or oligosaccharide having anN-acetylglucosamine unit at the reducing end and (ii) one or morelactose-based mammalian milk oligosaccharides (MMOs), wherein the cellis capable of: (i) synthesizing a nucleotide-sugar and themonosaccharide N-acetylglucosamine (GlcNAc) and (ii) expressing aglycosyltransferase to glycosylate the GlcNAc monosaccharide to producethe di- or oligosaccharide having an N-acetylglucosamine unit at thereducing end.

59. Cell according to any one of specific embodiments 56 or 58, whereinthe cell is metabolically engineered for the production of the di- oroligosaccharide having an N-acetylglucosamine at the reducing end.

60. Cell according to specific embodiment 57, wherein the cell ismetabolically engineered for the production of the oligosaccharidehaving an N-acetylglucosamine at the reducing end.

61. Cell according to any one of specific embodiments 56 to 60, whereinthe cell is modified with one or more gene expression modules,characterized in that the expression from any of the expression modulesis either constitutive or is created by a natural inducer.

62. Cell according to any one of specific embodiments 56 to 61, whereinthe cell comprises multiple copies of the same coding DNA sequenceencoding for one protein.

63. Cell according to any one of specific embodiments 56 to 62, whereinthe cell expresses at least one glucosamine 6-phosphateN-acetyltransferase and a phosphatase to synthesize N-acetylglucosamine.

64. Cell according to any one of specific embodiments 56 to 63, whereinthe cell expresses at least one glycosyltransferase to glycosylateN-acetylglucosamine.

65. Cell according to any one of specific embodiments 56 to 64, whereinthe cell is modified in the expression or activity of an enzyme selectedfrom the group comprising: glucosamine 6-phosphate N-acetyltransferase,phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.

66. Cell according to any one of specific embodiments 56 to 65, whereinthe nucleotide-sugar is selected from the group comprising:UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc),UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine(UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose(UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, and UDP-xylose.

67. Cell according to any one of specific embodiments 56 to 66, whereinthe nucleotide-sugar is UDP-galactose and the glycosyltransferase is anN-acetylglucosamine b-1,3-galactosyltransferase or anN-acetylglucosamine b-1,4-galactosyltransferase.

68. Cell according to any one of specific embodiments 56 to 67, whereinthe oligosaccharide has a lacto-N-biose (Gal-b1,3-GlcNAc) or anN-acetyllactosamine (Gal-b1,4-GlcNAc) at the reducing end.

69. Cell according to any one of specific embodiments 56 to 68, whereinthe N-acetylglucosamine b-1,3-galactosyltransferase is aglycosyltransferase having

-   -   a. PFAM domain PF00535, and        -   i. comprises the sequence [AGPS]XXLN(X_(n))RXDXD with SEQ ID            NO:01, wherein X is any amino acid and wherein n is 12 to            17, or        -   ii. comprises the sequence            PXXLN(X_(n))RXDXD(X_(m))[FWY]XX[HKR]XX[NQST] with SEQ ID            NO:02, wherein X is any amino acid and wherein n is 12 to 17            and m 100 to 115, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NOs: 03, 04, 05, 06, 07 or 08, preferably of any            one of SEQ ID NO:03, 04, 05, 06 or 07, more preferably any            one of SEQ ID NO:03, 06 or 07, most preferably any one of            SEQ ID NO:03 or 06, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NOs: NOs: 03, 04, 05, 06, 07 or 08, preferably            of any one of SEQ ID NO:03, 04, 05, 06 or 07, more            preferably any one of SEQ ID NO:03, 06 or 07, most            preferably any one of SEQ ID NO:03 or 06, having at least            80% overall sequence identity to the full-length of any one            of the N-acetylglucosamine b-1,3-galactosyltransferase            polypeptide with SEQ ID NO: 03, 04, 05, 06, 07 or 08,            preferably of any one of SEQ ID NO:03, 04, 05, 06 or 07,            more preferably any one of SEQ ID NO:03, 06 or 07, most            preferably any one of SEQ ID NO:03 or 06, and having            N-acetylglucosamine b-1,3-galactosyltransferase activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 03, 04, 05, 06, 07            or 08, preferably of any one of SEQ ID NO:03, 04, 05, 06 or            07, more preferably any one of SEQ ID NO:03, 06 or 07, most            preferably any one of SEQ ID NO:03 or 06, and having            N-acetylglucosamine b-1,3-galactosyltransferase activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 03,            04, 05, 06, 07 or 08, preferably of any one of SEQ ID NO:03,            04, 05, 06 or 07, more preferably any one of SEQ ID NO:03,            06 or 07, most preferably any one of SEQ ID NO:03 or 06, and            having N-acetylglucosamine b-1,3-galactosyltransferase            activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            03, 04, 05, 06, 07 or 08, preferably of any one of SEQ ID            NO:03, 04, 05, 06 or 07, more preferably any one of SEQ ID            NO:03, 06 or 07, most preferably any one of SEQ ID NO:03 or            06, and having N-acetylglucosamine            b-1,3-galactosyltransferase activity, or    -   b. PFAM domain IPR002659, and        -   i. comprises the sequence            KT(X_(n))[FY]XXKXDXD(X_(m))[FHY]XXG(X, no A, G,            S)(X_(p))X(no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO:09,            wherein X is any amino acid and wherein n is 13 to 16, m 35            to 70 and p 20 to 45, or        -   ii. comprises a polypeptide sequence according to any one of            SEQ ID NOs: 10, 11, 12 or 13, or        -   iii. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 10, 11, 12 or 13having at least 80%            overall sequence identity to the full-length of any one of            the N-acetylglucosamine b-1,3-galactosyltransferase            polypeptide with SEQ ID NO: 10, 11, 12 or 13 and having            N-acetylglucosamine b-1,3-galactosyltransferase activity, or        -   iv. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 10, 11, 12 or 13            and having N-acetylglucosamine b-1,3-galactosyltransferase            activity, or        -   v. is a functional fragment of any one of SEQ ID NO: 10, 11,            12 or 13 and having N-acetylglucosamine            b-1,3-galactosyltransferase activity, or        -   vi. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            10, 11, 12 or 13 and having N-acetylglucosamine            b-1,3-galactosyltransferase activity.

70. Cell according to any one of specific embodiments 56 to 69, whereinthe N-acetylglucosamine b-1,4-galactosyltransferase is aglycosyltransferase having

-   -   a. PFAM domain PF01755, and        -   i. comprises the sequence            EXXCXXSHXX[ILV][FWY](X_(n))EDD(X_(m))[ACGST]XXYX[ILMV] with            SEQ ID NO:14, wherein X is any amino acid and wherein n is            13 to 15 and m 50 to 76, or        -   ii. comprises a polypeptide sequence according to any one of            SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23, preferably            of any one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more            preferably of any one of SEQ ID NO: 17, 18, 20 or 21, or        -   iii. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22 or 23,            preferably of any one of SEQ ID NO: 15, 16, 17, 18, 20 or            21, more preferably of any one of SEQ ID NO: 17, 18, 20 or            21, having at least 80% overall sequence identity to the            full-length of any one of the N-acetylglucosamine            b-1,4-galactosyltransferase polypeptide with SEQ ID NO: 15,            16, 17, 18, 19, 20, 21, 22 or 23, preferably of any one of            SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably of any            one of SEQ ID NO: 17, 18, 20 or 21, and having            N-acetylglucosamine b-1,4-galactosyltransferase activity, or        -   iv. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 15, 16, 17, 18, 19,            20, 21, 22 or 23, preferably of any one of SEQ ID NO: 15,            16, 17, 18, 20 or 21, more preferably of any one of SEQ ID            NO: 17, 18, 20 or 21, and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v. is a functional fragment of any one of SEQ ID NO: 15, 16,            17, 18, 19, 20, 21, 22 or 23, preferably of any one of SEQ            ID NO: 15, 16, 17, 18, 20 or 21, more preferably of any one            of SEQ ID NO: 17, 18, 20 or 21, and having            N-acetylglucosamine b-1,4-galactosyltransferase activity, or        -   vi. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            15, 16, 17, 18, 19, 20, 21, 22 and or 23, preferably of any            one of SEQ ID NO: 15, 16, 17, 18, 20 or 21, more preferably            of any one of SEQ ID NO: 17, 18, 20 or 21, and having            N-acetylglucosamine b-1,4-galactosyltransferase activity, or    -   b. PFAM domain PF00535, and        -   i. comprises the sequence            R[KN]XXXXXXXGXXXX[FL]XDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE]            with SEQ ID NO:24, wherein X is any amino acid and wherein n            is 50 to 75 and m 10 to 30, or        -   ii. comprises the sequence            R[KN]XXXXXXXGXXXXFXDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE](X_(p))            [FWY]XX[HKR]XX[NQST] with SEQ ID NO:25, wherein X is any            amino acid and wherein n is 50 to 75, m is 10 to 30 and p is            20 to 25, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NO: 26, 27 or 28, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 26, 27 or 28 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 26, 27 or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 26, 27 or 28 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 26, 27            or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            26, 27 or 28 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   c. PFAM domain PF02709 and not having PFAM domain PF00535,        -   i. comprises the sequence            [FWY]XX[FY][FWY](X₂₃)[FWY][GQ]X[DE]D with SEQ ID NO:29,            wherein X is any amino acid, or        -   ii. comprises the sequence [PV]W[GHNP](X_(n))[FWY][GQ]X[DE]D            with SEQ ID NO:30, wherein X is any amino acid and wherein n            is 21 to 24, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NO: 31, 32, 33, 34, 35 or 36, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 31, 32, 33, 34, 35 or 36 having at least            80% overall sequence identity to the full-length of any one            of the N-acetylglucosamine b-1,4-galactosyltransferase            polypeptide with SEQ ID NO: 31, 32, 33, 34, 35 or 36 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO: 31, 32, 33, 34, 35            or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 31,            32, 33, 34, 35 or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID NO:            31, 32, 33, 34, 35 or 36 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or    -   d. PFAM domain PF03808, and        -   i. comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA with SEQ ID            NO:37, wherein X is any amino acid and wherein n is 20 to            25, or        -   ii. comprises the sequence            [ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA(X_(m))[HR]XG[FWY](X_(p))GXGXXXQ[DE]            with SEQ ID NO:38, wherein X is any amino acid and wherein n            is 20 to 25, m is 40 to 50 and p is 22 to 30, or        -   iii. comprises a polypeptide sequence according to any one            of SEQ ID NO: 39, 40 or 41, or        -   iv. is a functional homologue, variant or derivative of any            one of SEQ ID NO: 39, 40 or 41 having at least 80% overall            sequence identity to the full-length of any one of the            N-acetylglucosamine b-1,4-galactosyltransferase polypeptide            with SEQ ID NO: 39, 40 or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   v. comprises an oligopeptide sequence of at least 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino            acid residues from any one of SEQ ID NO:39, 40 or 41 and            having N-acetylglucosamine b-1,4-galactosyltransferase            activity, or        -   vi. is a functional fragment of any one of SEQ ID NO: 39, 40            or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity, or        -   vii. comprises a polypeptide comprising or consisting of an            amino acid sequence having at least 80% sequence identity to            the full-length amino acid sequence of any one of SEQ ID            NO:39, 40 or 41 and having N-acetylglucosamine            b-1,4-galactosyltransferase activity.

71. The cell according to any one of specific embodiments 56 to 70,wherein

-   -   a. the glucosamine 6-phosphate N-acetyltransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P43577 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P43577 having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P43577 and having glucosamine 6-phosphate        N-acetyltransferase activity, and    -   b. the L-glutamine-D-fructose-6-phosphate aminotransferase is a        polypeptide sequence comprising the polypeptide with UniProt ID        P17169 or is a functional homologue, variant or derivative of        the polypeptide with UniProt ID P17169having at least 80%        overall sequence identity to the full-length of the polypeptide        with UniProt ID P17169 and having        L-glutamine-D-fructose-6-phosphate aminotransferase activity; or        is a modified version that differs from the polypeptide with        UniProt ID P17169by an A39T, an R250C and an G472S mutation.

72. The cell according to any one of specific embodiments 56 to 71,wherein the cell is capable to catabolize a carbon source selected fromthe list comprising: glucose, fructose, galactose, lactose, sucrose,maltose, malto-oligosaccharides, maltotriose, sorbitol, xylose,rhamnose, mannose, methanol, ethanol, arabinose, trehalose, starch,cellulose, hemi-cellulose, corn-steep liquor, high-fructose syrup,molasses, glycerol, acetate, citrate, lactate, pyruvate.

73. The cell according to any one of specific embodiments 56 to 72,wherein the cell is unable to convert N-acetylglucosamine-6-phosphate toglucosamine-6-phosphate, and/or unable to convertglucosamine-6-phosphate to fructose-6-phoshate.

74. The cell according to any one of specific embodiments 56 to 73,wherein the cell is modified to produce GDP-fucose.

75. The cell according to any one of specific embodiments 56 to 74,wherein the cell is modified for enhanced GDP-fucose production andwherein the modification is chosen from the group comprising: knock-outof an UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferaseencoding gene, over-expression of a GDP-L-fucose synthase encoding gene,over-expression of a GDP-mannose 4,6-dehydratase encoding gene,over-expression of a mannose-1-phosphate guanylyltransferase encodinggene, over-expression of a phosphomannomutase encoding gene orover-expression of a mannose-6-phosphate isomerase encoding gene.

76. The cell according to any one of specific embodiments 56 to 75,wherein the cell is modified to produce UDP-galactose.

77. The cell according to any one of specific embodiments 56 to 76,wherein the cell is modified for enhanced UDP-galactose production andwherein the modification is chosen from the group comprising: knock-outof an 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out ofan galactose-1-phosphate uridylyltransferase encoding gene.

78. The cell according to any one of specific embodiments 56 to 75,wherein the cell is modified to produce CMP-N-acetylneuraminic acid.

79. The cell according to any one of specific embodiments 56 to 78,wherein the cell is modified for enhanced CMP-N-acetylneuraminic acidproduction and wherein the modification is chosen from the groupcomprising: over-expression of an CMP-sialic acid synthetase encodinggene, over-expression of a sialate synthase encoding gene,over-expression of an N-acetyl-D-glucosamine 2-epimerase encoding gene.

80. The cell according to any one of specific embodiments 56 to 79,wherein the cell is capable to express least one furtherglycosyltransferase and wherein the further glycosyltransferase ischosen from the group comprising: fucosyltransferases,sialyltransferases, galactosyltransferases, glucosyltransferases,mannosyltransferases, N-acetylglucosaminyltransferases,N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases,xylosyltransferases, glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases, N-acetylrhamnosyltransferases,UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases,UDP-N-acetylglucosamine enolpyruvyl transferases andfucosaminyltransferases,

-   -   preferably, the fucosyltransferase is chosen from the list        comprising alpha-1,2-fucosyltransferase,        alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase and        alpha-1,6-fucosyltransferase,    -   preferably, the sialyltransferase is chosen from the list        comprising alpha-2,3-sialyltransferase,        alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase,    -   preferably, the galactosyltransferase is chosen from the list        comprising beta-1,3-galactosyltransferase, N-acetylglucosamine        beta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase,        N-acetylglucosamine beta-1,4-galactosyltransferase,        alpha-1,3-galactosyltransferase and        alpha-1,4-galactosyltransferase,    -   preferably, the glucosyltransferase is chosen from the list        comprising alpha-glucosyltransferase,        beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and        beta-1,4-glucosyltransferase,    -   preferably, the mannosyltransferase is chosen from the list        comprising alpha-1,2-mannosyltransferase,        alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase,    -   preferably, the N-acetylglucosaminyltransferase is chosen from        the list comprising galactoside        beta-1,3-N-acetylglucosaminyltransferase and        beta-1,6-N-acetylglucosaminyitransferase,    -   preferably, the N-acetylgalactosaminyltransferase is an        alpha-1,3-N-acetylgalactosaminyltransferase.

81. The cell according to specific embodiment 80, wherein the cell ismodified in the expression or activity of the furtherglycosyltransferase.

82. The cell according to any one of specific embodiments 56 to 81,wherein the cell is using one or more precursor(s) for the production ofthe di- or oligosaccharide having a GlcNAc unit at the reducing end, theprecursor(s) being fed to the cell from the cultivation medium.

83. The cell according to any one of specific embodiments 56 to 82,wherein the cell is producing one or more precursor(s) for theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

84. The cell according to any one of specific embodiments 82 or 83,wherein the precursor for the production of the di- or oligosaccharideis completely converted into the di- or oligosaccharide having a GlcNAcunit at the reducing end.

85. The cell according to any one of specific embodiments 56 to 84,wherein the cell produces the di- or oligosaccharide having a GlcNAcunit at the reducing end intracellularly and wherein a fraction orsubstantially all of the produced di- or oligosaccharide having a GlcNAcunit at the reducing end remains intracellularly and/or is excretedoutside the cell via passive or active transport.

86. The cell according to any one of specific embodiments 56 to 85,wherein the cell expresses a membrane transporter protein or apolypeptide having transport activity hereby transporting compoundsacross the outer membrane of the cell wall,

-   -   preferably, the cell is modified in the expression or activity        of the membrane transporter protein or polypeptide having        transport activity.

87. The cell according to specific embodiment 86, wherein the membranetransporter protein or polypeptide having transport activity is chosenfrom the list comprising porters, P—P-bond-hydrolysis-driventransporters, β-barrel porins, auxiliary transport proteins, putativetransport proteins and phosphotransfer-driven group translocators,

-   -   preferably, the porters comprise MFS transporters, sugar efflux        transporters and siderophore exporters,    -   preferably, the P—P-bond-hydrolysis-driven transporters comprise        ABC transporters and siderophore exporters.

88. The cell according to any one of specific embodiments 86 or 87,wherein the membrane transporter protein or polypeptide having transportactivity controls the flow over the outer membrane of the cell wall ofthe di- or oligosaccharide having a GlcNAc unit at the reducing endand/or of one or more precursor(s) and/or acceptor(s) to be used in theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

89. The cell according to any one of specific embodiments 86 to 88,wherein the membrane transporter protein or polypeptide having transportactivity provides improved production and/or enabled and/or enhancedefflux of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

90. The cell according to any one of the specific embodiments 56 to 89,wherein the cell comprises a modification for reduced production ofacetate compared to a non-modified progenitor.

91. The cell according to specific embodiment 90, wherein the cellcomprises a lower or reduced expression and/or abolished, impaired,reduced or delayed activity of any one or more of the proteinscomprising beta-galactosidase, galactoside O-acetyltransferase,N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphatedeaminase, N-acetylglucosamine repressor, ribonucleotidemonophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase,N-acetylneuraminate lyase, N-acetylmannosamine kinase,N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man,EIID-Man, ushA, galactose-1-phosphate uridylyltransferase,glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase,ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobicrespiration control protein, transcriptional repressor IclR, lonprotease, glucose-specific translocating phosphotransferase enzyme IIBCcomponent ptsG, glucose-specific translocating phosphotransferase (PTS)enzyme IIBC component malX, enzyme IIA^(Glc), beta-glucoside specificPTS enzyme II, fructose-specific PTS multiphosphoryl transfer proteinFruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase,pyruvate-formate lyase, acetate kinase, phosphoacyltransferase,phosphate acetyltransferase, pyruvate decarboxylase compared to anon-modified progenitor.

92. The cell according to any one of the specific embodiments 56 to 91,wherein the cell is capable to produce phosphoenolpyruvate (PEP).

93. The cell according to any one of the specific embodiments 56 to 92,wherein the cell is modified for enhanced production and/or supply ofphosphoenolpyruvate (PEP) compared to a non-modified progenitor.

94. The cell according to any one of the specific embodiments 56 to 93,wherein the cell comprises a catabolic pathway for selected mono-, di-or oligosaccharides which is at least partially inactivated, the mono-,di-, or oligosaccharides being involved in and/or required for theproduction of the di- or oligosaccharide having a GlcNAc unit at thereducing end.

95. The cell according to any one of the specific embodiments 56 to 94,wherein the cell resists the phenomenon of lactose killing when grown inan environment in which lactose is combined with one or more othercarbon source(s).

96. The cell according to any one of the specific embodiments 56 to 95,wherein the cell produces 90 g/L or more of the di- or oligosaccharidehaving a GlcNAc at the reducing end in the whole broth and/orsupernatant and/or wherein the di- or oligosaccharide having a GlcNAc atthe reducing end in the whole broth and/or supernatant has a purity ofat least 80% measured on the total amount of the di- or oligosaccharidehaving a GlcNAc unit at the reducing end and its precursor(s) in thewhole broth and/or supernatant, respectively.

97. The cell according to any one of the specific embodiments 56 to 96,wherein the cell produces a mixture of charged, preferably sialylated,and/or neutral di- and oligosaccharides comprising at least one di- oroligosaccharide having a GlcNAc unit at the reducing end.

98. The cell according to any one of the specific embodiments 56 to 97,wherein the cell produces a mixture of charged, preferably sialylated,and/or neutral oligosaccharides comprising at least one oligosaccharidehaving a GlcNAc unit at the reducing end.

99. The cell according to any one of specific embodiments 56 to 98,wherein the oligosaccharide is chosen from the list comprising:2-fucosyl lacto-N-biose, 4-fucosyl lacto-N-biose, 2-4-difucosyllacto-N-biose, 3′-sialyl lacto-N-biose, 6′-sialyl lacto-N-biose,3′,6′-disialyl lacto-N-biose, 6,6′-disialyl lacto-N-biose,2′-fucosyl-3′-sialyl lacto-N-biose, 2′-fucosyl-6′-sialyl lacto-N-biose,4-fucosyl-3′-sialyl lacto-N-biose, 4-fucosyl-6′-sialyl lacto-N-biose,2-fucosyl N-acetyllactosamine, 3′-fucosyl N-acetyllactosamine,2,3′-difucosyl N-acetyllactosamine, 3′-sialyl N-acetyllactosamine,6′-sialyl N-acetyllactosamine, 3′,6′-disialyl N-acetyllactosamine,6,6′-disialyl N-acetyllactosamine, 2′-fucosyl-3′-sialylN-acetyllactosamine, 2′-fucosyl-6′-sialyl N-acetyllactosamine,3-fucosyl-3′-sialyl N-acetyllactosamine, 3′-fucosyl-6′-sialylN-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), thexenotransplantation epitope (Gal-a1,3-Gal-b1,4-GlcNAc),Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine,GalNAc-b1,3-Gal-b1,4-GlcNAc.

100. The cell according to any one of the specific embodiments 56 or 58to 99, wherein the disaccharide having a GlcNAc unit at the reducing enddoes not comprise chitobiose (GlcNAc-GlcNAc).

101. The cell according to any one of the specific embodiments 56 to100, wherein the oligosaccharide having a GlcNAc unit at the reducingend does not comprise a chitobiose at the reducing end, preferably doesnot comprise an N-glycan.

102. The cell according to any one of specific embodiments 56 to 101 ormethod according to any one of specific embodiments 1 to 55, wherein thecell is a bacterium, fungus, yeast, a plant cell, an animal cell, or aprotozoan cell,

-   -   preferably the bacterium is an Escherichia coli strain, more        preferably an Escherichia coli strain which is a K-12 strain,        even more preferably the Escherichia coli K-12 strain is E. coli        MG1655,    -   preferably the fungus belongs to a genus chosen from the group        comprising Rhizopus, Dictyostelium, Penicillium, Mucor or        Aspergillus,    -   preferably the yeast belongs to a genus chosen from the group        comprising Saccharomyces, Zygosaccharomyces, Pichia,        Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or        Debaromyces,    -   preferably the plant cell is an algal cell or is derived from        tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or        corn plant,    -   preferably the animal cell is derived from non-human mammals,        birds, fish, invertebrates, reptiles, amphibians or insects or        is a genetically modified cell line derived from human cells        excluding embryonic stem cells, more preferably the human and        non-human mammalian cell is an epithelial cell, an embryonic        kidney cell, a fibroblast cell, a COS cell, a Chinese hamster        ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a        non-mammary adult stem cell or derivatives thereof, more        preferably the insect cell is derived from Spodoptera        frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or        Drosophila melanogaster,    -   preferably the protozoan cell is a Leishmania tarentolae cell.

103. The cell according to specific embodiment 102 or method accordingto specific embodiment 102, wherein the cell is a viable Gram-negativebacterium that comprises a reduced or abolished synthesis ofpoly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA),cellulose, colanic acid, core oligosaccharides, OsmoregulatedPeriplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalosecompared to a non-modified progenitor.

104. The method according to any one of specific embodiments 1 to 55,102 or 103, wherein the separation comprises at least one of thefollowing steps: clarification, ultrafiltration, nanofiltration,two-phase partitioning, reverse osmosis, microfiltration, activatedcharcoal or carbon treatment, treatment with non-ionic surfactants,enzymatic digestion, tangential flow high-performance filtration,tangential flow ultrafiltration, affinity chromatography, ion exchangechromatography, hydrophobic interaction chromatography and/or gelfiltration, ligand exchange chromatography.

105. The method according to any one of specific embodiments 1 to 55,102 to 104, further comprising purification of the di- oroligosaccharide from the cell.

106. The method according to any one of specific embodiments 1 to 55,102 to 105, wherein the purification comprises at least one of thefollowing steps: use of activated charcoal or carbon, use of charcoal,nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment orion exchange, use of alcohols, use of aqueous alcohol mixtures,crystallization, evaporation, precipitation, drying, spray drying,lyophilization, spray freeze drying, freeze spray drying, band drying,belt drying, vacuum band drying, vacuum belt drying, drum drying, rollerdrying, vacuum drum drying or vacuum roller drying.

107. Use of a cell according to any one of specific embodiments 56 to103, or a method according to any one of specific embodiments 1 to 55,102 to 106 for the production of a di- or oligosaccharide having anN-acetylglucosamine unit at the reducing end.

108. Use of a cell according to any one of specific embodiments 56, 58to 103 or a method according to any one of specific embodiments 1, 3 to55, 102 to 106 for the production of an oligosaccharide having anN-acetylglucosamine unit at the reducing end, preferably for theproduction of a charged or neutral oligosaccharide having anN-acetylglucosamine unit at the reducing end, more preferably for theproduction of a sialylated or fucosylated form of LNB or LacNAc.

The following examples will serve as further illustration andclarification of the disclosure and are not intended to be limiting.

EXAMPLES Example 1: Materials and Methods Escherichia coli

Media

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco,Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodiumchloride (VWR. Leuven, Belgium). The medium used in the cultivationexperiments in 96-well plates or in shake flasks contained 2.00 g/LNH₄Cl, 5.00 g/L (NH₄)₂SO₄, 2.993 g/L KH₂PO₄, 7.315 g/L K2HPO₄, 8.372 g/LMOPS, 0.5 g/L NaCl, 0.5 g/L MgSO₄·7H₂O, 30 g/L sucrose or 30 g/Lglycerol, 1 ml/L vitamin solution, 100 μl/L molybdate solution, and 1mL/L selenium solution. As specified in the respective examples, 0.30g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB wereadditionally added to the medium as precursor(s). The medium was set toa pH of 7 with 1M KOH. Vitamin solution consisted of 3.6 g/L FeCl₂·4H₂O,5 g/L CaCl₂·2H₂O, 1.3 g/L MnCl₂·2H₂O, 0.38 g/L CuCl₂·2H₂O, 0.5 g/LCoCl₂·6H₂O, 0.94 g/L ZnCl₂, 0.0311 g/L H₃BO₄, 0.4 g/L Na₂EDTA·2H₂O and1.01 g/L thiamine·HCl. The molybdate solution contained 0.967 g/LNaMoO₄·2H₂O. The selenium solution contained 42 g/L SeO₂.

The minimal medium for fermentations contained 6.75 g/L NH₄Cl, 1.25 g/L(NH₄)₂SO₄, 2.93 g/L KH₂PO₄ and 7.31 g/L KH₂PO₄, 0.5 g/L NaCl, 0.5 g/LMgSO₄·7H₂O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution,100 μL/L molybdate solution, and 1 mL/L selenium solution with the samecomposition as described above. As specified in the respective examples,0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNBwere additionally added to the minimal medium for fermentations asprecursor(s).

Complex medium was sterilized by autoclaving (121° C., 21 min) andminimal medium by filtration (0.22 μm Sartorius). When necessary, themedium was made selective by adding an antibiotic: e.g., chloramphenicol(20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/orkanamycin (50 mg/L).

Plasmids

pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains anFRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains anFRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLPrecombinase activity) plasmids were obtained from Prof. R. Cunin (VrijeUniversiteit Brussel, Belgium in 2007).

Plasmids were maintained in the host E. coli DH5alpha (F⁻,phi80dlacZΔM15, Δ(iacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk⁻,mk⁺), phoA, supE44, lambda, thi-1, gyrA96, relA1) bought fromInvitrogen.

Strains and Mutations

Escherichia coli K12 MG1655 [λ⁻, F⁻, rph-1] was obtained from the ColiGenetic Stock Center (US), CGSC Strain #: 7740, in March 2007. Genedisruptions, gene introductions and gene replacements were performedusing the technique published by Datsenko and Wanner (PNAS 97 (2000),6640-6645). This technique is based on antibiotic selection afterhomologous recombination performed by lambda Red recombinase. Subsequentcatalysis of a flippase recombinase ensures removal of the antibioticselection cassette in the final production strain.

Transformants carrying a Red helper plasmid pKD46 were grown in 10 mL LBmedia with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30° C. toan OD₆₀₀ nm of 0.6. The cells were made electrocompetent by washing themwith 50 mL of ice-cold water, a first time, and with 1 mL ice coldwater, a second time. Then, the cells were resuspended in 50 μL ofice-cold water. Electroporation was done with 50 μL of cells and 10-100ng of linear double-stranded-DNA product by using a Gene Pulser™(BioRad) (600 Ω, 25 μFD and 250 volts).

After electroporation, cells were added to 1 mL LB media incubated 1 hat 37° C., and finally spread onto LB-agar containing 25 mg/L ofchloramphenicol or 50 mg/L of kanamycin to select antibiotic resistanttransformants. The selected mutants were verified by PCR with primersupstream and downstream of the modified region and were grown in LB-agarat 42° C. for the loss of the helper plasmid. The mutants were testedfor ampicillin sensitivity.

The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 andtheir derivates as template. The primers used had a part of the sequencecomplementary to the template and another part complementary to the sideon the chromosomal DNA where the recombination must take place. For thegenomic knock-out, the region of homology was designed 50-nt upstreamand 50-nt downstream of the start and stop codon of the gene ofinterest. For the genomic knock-in, the transcriptional starting point(+1) had to be respected. PCR products were PCR-purified, digested withDpnl, re-purified from an agarose gel, and suspended in elution buffer(5 mM Tris, pH 8.0).

Selected mutants were transformed with pCP20 plasmid, which is anampicillin and chloramphenicol resistant plasmid that showstemperature-sensitive replication and thermal induction of FLPsynthesis. The ampicillin-resistant transformants were selected at 30°C., after which a few were colony purified in LB at 42° C. and thentested for loss of all antibiotic resistance and of the FLP helperplasmid. The gene knock-outs and knock-ins are checked with controlprimers.

In one example for GDP-fucose and fucosylated oligosaccharideproduction, the mutant strain was derived from E. coli K12 MG1655comprising knock-outs of the E. coli wcaJ and thyA genes and genomicknock-ins of constitutive expression constructs containing a sucrosetransporter like e.g., CscB originating from E. coli W (UniProt IDE0IXR1), a fructose kinase like e.g., frk originating from Zymomonasmobilis (ZmFrk) (UniProt ID Q03417), a sucrose phosphorylase like e.g.,BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6),additionally comprising expression plasmids with constitutive expressionconstructs for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H.pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferaselike e.g., HpFucT from H. pylori (UniProt ID O30511) and with aconstitutive expression construct for the E. coli thyA (UniProt IDP0A884) as selective marker. The constitutive expression constructs ofthe fucosyltransferase genes can also be present in the mutant E. colistrain via genomic knock-ins. GDP-fucose production can further beoptimized in the mutant E. coli strain by genomic knock-outs of the E.coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi andion as described in WO 2016075243 and WO 2012007481. GDP-fucoseproduction can additionally be optimized comprising genomic knock-ins ofconstitutive expression constructs for a mannose-6-phosphate isomeraseslike e.g., manA from E. coli (UniProt ID P00946), a phosphomannomutaselike e.g., manB from E. coli (UniProt ID P24175), a mannose-1-phosphateguanylyltransferase like e.g., manC from E. coli (UniProt ID P24174), aGDP-mannose 4,6-dehydratase like e.g., gmd from E. coli (UniProt IDPOAC88) and a GDP-L-fucose synthase like e.g., fcl from E. coli (UniProtID P32055). GDP-fucose production can also be obtained by genomicknock-outs of the E. coli fucK and fucI genes together with genomicknock-ins of constitutive expression constructs containing a fucosepermease like e.g., fucP from E. coli (UniProt ID P11551) and abifunctional enzyme with fucose kinase/fucose-1-phosphateguanylyltransferase activity like e.g., fkp from Bacteroides fragilis(UniProt ID SUV40286.1). If the mutant strain producing GDP-fucose wasintended to make fucosylated lactose structures, the strain wasadditionally modified with genomic knock-outs of the E. coli LacZ, LacYand LacA genes and with a genomic knock-in of a constitutive expressionconstruct for a lactose permease like e.g., the E. coli LacY (UniProt IDP02920).Alternatively, and/or additionally, GDP-fucose and/orfucosylated oligosaccharide production can further be optimized in themutant E. coli strains with genomic knock-ins of constitutivetranscriptional units comprising a membrane transporter protein likee.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfAfrom Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProtID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceTfrom E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae(UniProt ID D4B8A6).

In one example for sialic acid production, the mutant strain was derivedfrom E. coli K12 MG1655 comprising genomic knock-ins of constitutivetranscriptional units containing one or more copies of a glucosamine6-phosphate N-acetyltransferase like e.g., GNA1 from Saccharomycescerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase likee.g., AGE from Bacteroides ovatus (UniProt ID A7LVG6) and anN-acetylneuraminate synthase like e.g., from Neisseria meningitidis(UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively, and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining an UDP-N-acetylglucosamine 2-epimerase like e.g., NeuC fromC. jejuni (UniProt ID Q93MP8) and an N-acetylneuraminate synthase likee.g., from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacterjejuni (UniProt ID Q93MP9).

Alternatively and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining a phosphoglucosamine mutase like e.g., glmM from E. coli(UniProt ID P31120), an N-acetylglucosamine-1-phosphateuridyltransferase/glucosamine-1-phosphate acetyltransferase like e.g.,glmU from E. coli (UniProt ID P0ACC7), an UDP-N-acetylglucosamine2-epimerase like e.g., NeuC from C. jejuni (UniProt ID Q93MP8) and anN-acetylneuraminate synthase like e.g., from Neisseria meningitidis(UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively, and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosaminekinase like e.g., from Mus musculus (strain C57BL/6J) (UniProt IDQ91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g., fromPseudomonas sp. UW4 (UniProt ID K9NPH9) and anN-acylneuraminate-9-phosphatase like e.g., from Candidatus Magnetomorumsp. HK-1 (UniProt ID KPA15328.1) or from Bacteroides thetaiotaomicron(UniProt ID Q8A712).

Alternatively, and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining a phosphoglucosamine mutase like e.g., glmM from E. coli(UniProt ID P31120), an N-acetylglucosamine-1-phosphateuridyltransferase/glucosamine-1-phosphate acetyltransferase like e.g.,glmU from E. coli (UniProt ID P0ACC7), a bifunctional UDP-GlcNAc2-epimerase/N-acetylmannosamine kinase like e.g., from M. musculus(strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphatesynthetase like e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9) andan N-acylneuraminate-9-phosphatase like e.g., from CandidatusMagnetomorum sp. HK-1 (UniProt ID KPA15328.1) or from Bacteroidesthetaiotaomicron (UniProt ID Q8A712).

Sialic acid production can further be optimized in the mutant E. colistrain with genomic knock-outs of the E. coli genes comprising any oneor more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manYand manZ as described in WO 2018122225, and/or genomic knock-outs of theE. coli genes comprising any one or more of nanT, poxB, ldhA, adhE,aldB, pflA, pflC, ybiY, ackA and/or pta and with genomic knock-ins ofconstitutive transcriptional units comprising one or more copies of anL-glutamine-D-fructose-6-phosphate aminotransferase like e.g., themutant glmS*54 from E. coli (differing from the wild-type E. coli glmS,having UniProt ID P17169, by an A39T, an R250C and an G472S mutation asdescribed by Deng et al. (Biochimie 88, 419-29 (2006)), preferably onephosphatase like any one or more of e.g., the E. coli genes comprisingaphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX,YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL,YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S.cerevisiae or BsAraL from Bacillus subtilis as described in WO2018122225 and an acetyl-CoA synthetase like e.g., acs from E. coli(UniProt ID P27550).

For sialylated oligosaccharide production, the sialic acid productionstrains were further modified to express an N-acylneuraminatecytidylyltransferase like e.g., the NeuA enzyme from C. jejuni (UniProtID Q93MP7), the NeuA enzyme from Haemophilus influenzae (GenBank No.AGV11798.1) or the NeuA enzyme from Pasteurella multocida (GenBank No.AMK07891.1) and to express one or more copies of a beta-galactosidealpha-2,3-sialyltransferase like e.g., PmultST3 from P. multocida(UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of aminoacid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactosidealpha-2,3-sialyltransferase activity, NmeniST3 from N. meningitidis(GenBank No. ARC07984.1) or PmultST2 from P. multocida subsp. multocidastr. Pm70 (GenBank No. AAK02592.1), a beta-galactosidealpha-2,6-sialyltransferase like e.g., PdST6 from Photobacteriumdamselae (UniProt ID O66375) or a PdST6-like polypeptide consisting ofamino acid residues 108 to 497 of UniProt ID O66375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt IDQ64689). Constitutive transcriptional units of the N-acylneuraminatecytidylyltransferase and the sialyltransferases can be delivered to themutant strain either via genomic knock-in or via expression plasmids. Ifthe mutant strains producing sialic acid and CMP-sialic acid wereintended to make sialylated lactose structures, the strains wereadditionally modified with genomic knock-outs of the E. coli LacZ, LacYand LacA genes and with a genomic knock-in of a constitutivetranscriptional unit for a lactose permease like e.g., E. coli LacY(UniProt ID P02920). All mutant strains producing sialic acid,CMP-sialic acid and/or sialylated oligosaccharides could optionally beadapted for growth on sucrose via genomic knock-ins of constitutivetranscriptional units containing a sucrose transporter like e.g., CscBfrom E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g., Frkoriginating from Z. mobilis (UniProt ID Q03417) and a sucrosephosphorylase like e.g., BaSP from B. adolescentis (UniProt ID A0ZZH6).

Alternatively, and/or additionally, sialic acid and/or sialylatedoligosaccharide production can further be optimized in the mutant E.coli strains with genomic knock-ins of constitutive transcriptionalunits comprising a membrane transporter protein like e.g., a sialic acidtransporter like e.g., nanT from E. coli K-12 MG1655 (UniProt IDP41036), nanT from E. coli O6:H1 (UniProt ID Q8FD59), nanT from E. coliO157:H7 (UniProt ID Q8X9G8) or nanT from E. albertii (UniProt ID B1EFH1) or a porter like e.g., EntS from E. coli (UniProt ID P24077),EntS from Kluyvera ascorbata (UniProt ID A0A378GQ13), EntS fromSalmonella enterica subsp. arizonae (UniProt ID A0A6Y2K4E8), MdfA fromCronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacteryoungae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfAfrom Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli(UniProt ID A0A024L207), iceT from Citrobacter youngae (UniProt IDD4B8A6), SetA from E. coli (UniProt ID P31675), SetB from E. coli(UniProt ID P33026) or SetC from E. coli (UniProt ID P31436) or an ABCtransporter like e.g., oppF from E. coli (UniProt ID P77737), lmrA fromLactococcus lactis subsp. lactis by. diacetylactis (UniProt IDA0A1V0NEL4), or Blon_2475 from Bifidobacterium longum subsp. infantis(UniProt ID B7GPD4).

In an example for enhanced UDP-galactose production, the E. coli K12MG1655 strain was modified with genomic knock-outs of any one or more ofthe E. coli ushA, galT, ldhA and agp genes and with a genomic knock-inof a constitutive expression construct for the UDP-glucose-4-epimerase(galE) of E. coli (UniProt ID P09147).

In an example for enhanced UDP-GlcNAc production, the E. coli K12 MG1655strain was modified with a genomic knock-in of a constitutivetranscriptional unit for an L-glutamine-D-fructose-6-phosphateaminotransferase like e.g., the mutant glmS*54 from E. coli (differingfrom the wild-type E. coli glmS protein, having UniProt ID P17169, by anA39T, an R250C and an G472S mutation as described by Deng et al.(Biochimie 2006, 88: 419-429).

In an example to produce lacto-N-triose (LN3, GlcNAc-b1,3-Gal-b1,4-Glc),the mutant strain was derived from E. coli K12 MG1655 and modified witha knock-out of the E. coli lacZ, lacY, lacA and nagB genes and withgenomic knock-ins of constitutive transcriptional units for a lactosepermease like e.g., the E. coli LacY (UniProt ID P02920) and agalactoside beta-1,3-N-acetylglucosaminyltransferase like e.g., lgtA(UniProt ID Q9JXQ6) from N. meningitidis.

In an example for production of LN3 derived oligosaccharides likelacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the mutantLN3 producing strain was further modified with a constitutivetranscriptional unit delivered to the strain either via genomic knock-inor from an expression plasmid for an N-acetylglucosaminebeta-1,3-galactosyltransferase like e.g., wbgO with SEQ ID NO:03 from E.coli O55:H₇.

In an example for production of LN3 derived oligosaccharides likelacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), themutant LN3 producing strain was further modified with a constitutivetranscriptional unit delivered to the strain either via genomic knock-inor from an expression plasmid for an N-acetylglucosaminebeta-1,4-galactosyltransferase like e.g., lgtB with SEQ ID NO:15 fromNeisseria meningitidis.

In an example for production of lacto-N-biose (LNB, Gal-b1,3-GlcNAc) andLNB-derived oligosaccharides the strains were modified with genomicknock-ins or expression plasmids comprising constitutive transcriptionalunits for one or more copies of a glucosamine 6-phosphateN-acetyltransferase like e.g., GNA1 from S. cerevisiae (UniProt IDP43577) and an N-acetylglucosamine beta-1,3-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 03, 04, 05, 06, 07, 08, 10, 11, 12and 13.

In an example for production of N-acetyllactosamine (LacNAc,Gal-b1,4-GlcNAc) and LacNAc-derived oligosaccharides the strains weremodified with genomic knock-ins or expression plasmids comprisingconstitutive transcriptional units for one or more copies of aglucosamine 6-phosphate N-acetyltransferase like e.g., GNA1 from S.cerevisiae (UniProt ID P43577) and an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40 or 41.

The mutant LNB, LacNAc, LN3, LNT and LNnT producing E. coli strains canalso optionally be adapted for growth on sucrose via genomic knock-insof constitutive transcriptional units containing a sucrose transporterlike e.g., CscB from E. coli W (UniProt ID E0IXR1), a fructose kinaselike e.g., Frk originating from Zymomonas mobilis (UniProt ID Q03417)and a sucrose phosphorylase like e.g., BaSP originating fromBifidobacterium adolescentis (UniProt ID A0ZZH6).

Alternatively, and/or additionally, production of LN3, LNT, LNnT, LNB,LacNAc and oligosaccharides derived thereof can further be optimized inthe mutant E. coli strains with genomic knock-ins of a constitutivetranscriptional unit comprising a membrane transporter protein likee.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfAfrom Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProtID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceTfrom E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae(UniProt ID D4B8A6).

Preferably but not necessarily, any one or more of theglycosyltransferases, the proteins involved in nucleotide-activatedsugar synthesis and/or membrane transporter protein were N- and/orC-terminally fused to a solubility enhancer tag like e.g., a SUMO-tag,an MBP-tag, His, FLAG®, Strep-II, Halo-tag, NusA, thioredoxin, GSTand/or the Fh8-tag to enhance their solubility (Costa et al., Front.Microbiol. 2014, doi.org/10.3389/fmicb.2014.00063; Fox et al., ProteinSci. 2001, 10(3), 622-630; Jia and Jeaon, Open Biol. 2016, 6: 160196).

Optionally, the mutant E. coli strains were modified with a genomicknock-in of a constitutive transcriptional unit encoding a chaperoneprotein like e.g., DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system(Baneyx F., Palumbo J. L. (2003) Improving Heterologous Protein Foldingvia Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P.E. (eds) E. coli Gene Expression Protocols. Methods in MolecularBiology™, vol 205. Humana Press).

Optionally, the mutant E. coli strains are modified to create aglycominimized E. coli strain comprising genomic knock-out of any one ormore of non-essential glycosyltransferase genes comprising pgaC, pgaD,rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL,waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl,arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA andyaiP.

All constitutive promoters and UTRs originated from the librariesdescribed by De Mey et al. (BMC Biotechnology, 2007), Dunn et al.(Nucleic Acids Res. 1980, 8(10), 2119-2132), Kim and Lee (FEBS letters1997, 407(3), 353-356) and Mutalik et al. (Nat. Methods 2013, No. 10,354-360).

The SEQ ID NOs: described in disclosure are summarized in Table 1.

All genes were ordered synthetically at Twist Bioscience(twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage wasadapted using the tools of the supplier.

All strains are stored in cryovials at −80° C. (overnight LB culturemixed in a 1:1 ratio with 70% glycerol).

TABLE 1 Overview of SEQ ID NOs: described in the disclosure SEQ Countryof origin of ID digital sequence NO: Organism Origin information 01 N.A.Synthetic Artificial sequence 02 N.A. Synthetic Artificial sequence 03E. coli O55:H7 Synthetic Germany 04 Pseudogulbenkiania Synthetic USAferrooxidans 05 Salmonella enterica Synthetic Australia subsp. salamaeserovar 06 Corynebacterium glutamicum Synthetic Japan 07 Photobacteriumleiognathi Synthetic USA 08 Chromobacterium violaceum Synthetic USA 09N.A. Synthetic Artificial sequence 10 A. thaliana Synthetic USA 11Trypanosoma brucei Synthetic Scotland (UK) 12 Mus musculus Synthetic USA13 Homo sapiens Synthetic Unknown 14 N.A. Synthetic Artificial sequence15 Neisseria meningitidis MC58 Synthetic United Kingdom 16 Neisseriameningitidis MC58 Synthetic United Kingdom 17 Helicobacter pyloriSynthetic Australia 18 Helicobacter pylori Synthetic Australia 19Aggregatibacter aphrophilus Synthetic United Kingdom 20 Helicobacterpylori Synthetic Australia 21 Pasteurella multocida Synthetic Australia22 Haemophilus influenzae Synthetic USA 23 Kingella denitrificansSynthetic Unknown 24 N.A. Synthetic Artificial sequence 25 N.A.Synthetic Artificial sequence 26 Streptococcus pneumoniae Synthetic USA27 Streptococcus agalactiae Synthetic United Kingdom 28 Hafnia alveiSynthetic Unknown 29 N.A. Synthetic Artificial sequence 30 N.A.Synthetic Artificial sequence 31 Steroidobacter denitrificans SyntheticGermany 32 Parachlamydiaceae Synthetic Japan bacterium HS-T3 33 Coxiellasp. DG_40 Synthetic USA 34 Bacteroides fragilis Synthetic United Kingdom35 Corallococcus exercitus Synthetic United Kingdom 36 Hypericibacteradhaerens Synthetic Germany 37 N.A. Synthetic Artificial sequence 38N.A. Synthetic Artificial sequence 39 Bacteroides vulgatus SyntheticUnited Kingdom 40 Prevotella copri Synthetic USA 41 PseudomonasSynthetic USA fluorescens 90F12-2

Cultivation Conditions

A preculture of 96-well microtiter plate experiments was started from acryovial, in 150 μL LB and was incubated overnight at 37° C. on anorbital shaker at 800 rpm. This culture was used as inoculum for a96-well square microtiter plate, with 400 μL MMsf medium by diluting400x. These final 96-well culture plates were then incubated at 37° C.on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. Tomeasure sugar concentrations at the end of the cultivation experimentwhole broth samples were taken from each well by boiling the culturebroth for 15 min at 60° C. before spinning down the cells (=average ofintra- and extracellular sugar concentrations).

A preculture for the bioreactor was started from an entire 1 mL cryovialof a certain strain, inoculated in 250 mL or 500 mL of MMsf medium in a1 L or 2.5 L shake flask and incubated for 24 h at 37° C. on an orbitalshaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL inoculumin 2 L batch medium); the process was controlled by MFCS controlsoftware (Sartorius Stedim Biotech, Melsungen, Germany). Culturingcondition were set to 37° C., and maximal stirring; pressure gas flowrates were dependent on the strain and bioreactor. The pH was controlledat 6.8 using 0.5 M H₂SO₄ and 20% NH₄OH. The exhaust gas was cooled. 10%solution of silicone antifoaming agent was added when foaming raisedduring the fermentation.

Optical Density

Cell density of the cultures was frequently monitored by measuringoptical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgiumor with a Spark 10M microplate reader, Tecan, Switzerland).

Analytical Analysis

Standards such as but not limited to sucrose, glucose,N-acetylglucosamine, N-acetyllactosamine, lacto-N-biose, fucosylatedN-acetyllactosamine (2′FLAcNAc, 3-FlacNAc), fucosylated lacto-N-biose(2′FLNB, 4-FLNB), sialylated N-acetyllactosamine (3′ SLacNAc, 6′ SLacNAcwere purchased from Carbosynth (UK), Elicityl (France) and IsoSep(Sweden). Other compounds were analyzed with in-house made standards.

N-acetylglucosamine and N-acetyllactosamine were analyzed on a DionexHPAEC system with pulsed amperometric detection (PAD). A volume of 54,of sample was injected on a Dionex CarboPac PA1 column 4×250 mm with aDionex CarboPac PA1 guard column 4×50 mm. The column temperature was 20°C. The separation was performed using 3 eluents: A) deionized water B)200 mM Sodium hydroxide and C) 500 mM Sodium acetate. The elutionprofile was applied as follow: 0-10 min 50% A and 50% B; 10-18 min50-44% A and 50% B; 18-28 min 44% A and 50% B; 28-32 min 44-30.8% A and50% B; 32-39 min 30.8% A and 50% B; 39-40 min 30.8-2% A and 50% B; 40-43min 2% A and 50% B; 43-44 min 2-50% A and 50% B; 44-50 min 50% A and 50%B. Flow rate was 1.0 mL/min.

N-acetylglucosamine, N-acetyllactosamine, lacto-N-biose, fucosylatedN-acetyllactosamine and fucosylated LNB were analyzed on a WatersAcquity H-class UPLC with Evaporative Light Scattering Detector (ELSD)or a Refractive Index (RI) detection. A volume of 0.7 μL sample wasinjected on a Waters Acquity UPLC BEH Amide column (2.1×100 mm; 130 Å;1.7 μm) column with an Acquity UPLC BEH Amide VanGuard column, 130 Å,2.1×5 mm. The column temperature was 50° C. The mobile phase consists ofa ¼ water and % acetonitrile solution were 0.2% Triethylamine was added.The method was isocratic with a flow of 0.130 mL/min. The ELS detectorhad a drift tube temperature of 50° C. and N2 gas pressure was 50 psi,gain 200 and data rate is 10 pps. The temperature of the RI detector wasset at 35° C.

Sialylated N-acetyllactosamine and sialylated lacto-N-biose wereanalyzed with a Waters Acquity H-Class UPLC with Refractive Index (RI)detection. A volume of 0.5 μL sample was injected on a Waters AcquityUPLC BEH Amide column (2.1×100 mm with 1.7 μm particle size) with amobile phase containing 70 mL acetonitrile, 26 mL 150 mM ammoniumacetate in ultrapure water and 4 mL methanol with 0.05% pyrrolidineadded. The method was isocratic with a flow rate of 0.150 mL/min. Thetemperature of the column was set at 50° C.

Sugars at low concentrations (below 50 mg/L) were analyzed on a DionexHPAEC system with pulsed amperometric detection (PAD). A volume of 5 μLof sample was injected on a Dionex CarboPac PA200 column 4×250 mm with aDionex CarboPac PA200 guard column 4×50 mm. The column temperature wasset to 30° C. A gradient was used wherein eluent A was deionized water,wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was500 mM Sodium acetate. The oligosaccharides were separated in 60 minwhile maintaining a constant ratio of 25% of eluent B using thefollowing gradient: an initial isocratic step maintained for 10 min of75% of eluent A, an initial increase from 0 to 4% of eluent C over 8min, a second isocratic step maintained for 6 min of 71% of eluent A and4% of eluent C, a second increase from 4 to 12% of eluent C over 2.6min, a third isocratic step maintained for 3.4 min of 63% of eluent Aand 12% of eluent C and a third increase from 12 to 48% of eluent C over5 min. As a washing step 48% of eluent C was used for 3 min. For columnequilibration, the initial condition of 75% of eluent A and 0% of eluentC was restored in 1 min and maintained for 11 min. The applied flow was0.5 mL/min.

Normalization of the Data

For all types of cultivation conditions, data obtained from the mutantstrains was normalized against data obtained in identical cultivationconditions with reference strains.

Example 2: Production of GlcNAc in a Modified E. coli Host

In this example, a wild-type E. coli K-12 MG1655 can first be modifiedwith a knock-out of the homologous E. coliN-acetylglucosamine-6-phosphate deacetylase (nagA) gene and the E. coliglucosamine-6-phosphate deaminase (nagB) gene and then be furthertransformed with an expression plasmid comprising a constitutiveexpression cassette for the glucosamine 6-phosphate N-acetyltransferaseGNA1 from Saccharomyces cerevisiae (UniProt ID P43577). The thusobtained mutant E. coli strain produces GlcNAc in whole broth sampleswhen evaluated in a growth experiment, according to the cultureconditions in Example 1, in which the culture medium contains glycerol.

Example 3: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 2 can further be transformed with a second compatible expressionplasmid comprising a constitutive expression cassette for the mutatedvariant of the L-glutamine-D-fructose-6-phosphate aminotransferaseglmS*54 from E. coli differing from the wild-type glmS protein (UniProtID P17169) by an A39T, an R250C and an G4725 mutation as described byDeng et al. (Biochimie 2006: 88, 419-429). The novel strain alsoproduces GlcNAc in whole broth samples when evaluated in a growthexperiment, according to the culture conditions in Example 1, in whichthe culture medium contains glycerol, and the GlcNAc titres obtained inthis strain are higher than the GlcNAc titres obtained with the mutantstrain created in Example 2 and lacking the mutant glmS*54.

Example 4: Production of GlcNAc in a Modified E. coli Host

A wild-type E. coli K-12 MG1655 can be modified with a knock-out of theE. coli nagA and the nagB genes and a genomic knock-in of a constitutiveexpression cassette for GNA1 from S. cerevisiae (UniProt ID P43577). Thethus obtained mutant E. coli strain produces GlcNAc in whole brothsamples when evaluated in a growth experiment, according to the cultureconditions in Example 1, in which the culture medium contains glycerol.

Example 5: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 4 can further be transformed with an expression plasmidcomprising a constitutive expression cassette for glmS*54 from E. colidiffering from the wild-type glmS protein (UniProt ID P17169) by anA39T, an R250C and an G472S mutation as described by Deng et al.(Biochimie 2006: 88, 419-429). The novel strain also produces GlcNAc inwhole broth samples when evaluated in a growth experiment, according tothe culture conditions in Example 1 in which the culture medium containsglycerol, and the GlcNAc titres obtained in this strain are higher thanthe GlcNAc titres obtained with the mutant strain created in Example 4and lacking the mutant glmS*54.

Example 6: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 4 can further be transformed with a genomic knock-in comprisinga constitutive expression cassette for glmS*54 from E. coli differingfrom the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250Cand an G472S mutation as described by Deng et al. (Biochimie 2006: 88,419-429). The novel strain also produces GlcNAc in whole broth sampleswhen evaluated in a growth experiment, according to the cultureconditions in Example 1, in which the culture medium contains glycerol,and the GlcNAc titres obtained in this strain are higher than the GlcNActitres obtained with the mutant strain created in Example 4 and lackingthe mutant glmS*54.

Example 7: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 2 can further be transformed with a genomic knock-in comprisinga constitutive expression cassette for glmS*54 from E. coli differingfrom the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250Cand an G472S mutation as described by Deng et al. (Biochimie 2006: 88,419-429). The novel strain also produces GlcNAc in whole broth sampleswhen evaluated in a growth experiment, according to the cultureconditions in Example 1, in which the culture medium contains glycerol,and the GlcNAc titres obtained in this strain are higher than the GlcNActitres obtained with the mutant strain created in Example 2 and lackingthe mutant glmS*54.

Example 8: Production of LacNAc or LNB in a Modified E. coli Host

The mutant GlcNAc-producing E. coli K-12 MG1655 strains having a nagABKO and expressing GNA1 (UniProt ID P43577) with or without additionalexpression of the mutant glmS*54 differing from the wild-type glmSprotein (UniProt ID P17169) by an A39T, an R250C and an G472S mutationas described by Deng et al. (Biochimie 2006: 88, 419-429) as describedin Examples 2 and 4 to 7 can further be transformed with a plasmidcontaining a constitutive transcriptional unit to express either theN-acetylglucosamine β1,4-galactosyltransferase LgtB of Neisseriameningitidis with SEQ ID NO:15 or the N-acetylglucosamineβ1,3-galactosyltransferase WbgO from E. coli O55:H7 with SEQ ID NO:03.

Each of the novel strains expressing LgtB with SEQ ID NO:15 produceGlcNAc and LacNAc in whole broth samples when evaluated in a growthexperiment, according to the culture conditions in Example 1, in whichthe culture medium contains glycerol. The GlcNAc and LacNAc titres arehigher in the novel strain expressing SEQ ID NO:15 and also expressingthe mutant glmS*54 compared to the modified strain expressing SEQ IDNO:15 but lacking the mutant glmS*54.

Each of the novel strains expressing WbgO with SEQ ID NO:03 produceGlcNAc and LNB in whole broth samples when evaluated in a growthexperiment, according to the culture conditions in Example 1, in whichthe culture medium contains glycerol. The GlcNAc and LNB titres arehigher in the novel strain expressing SEQ ID NO:03 and also expressingthe mutant glmS*54 compared to the modified strain expressing WbgO withSEQ ID NO:03 but lacking the mutant glmS*54.

Example 9: Production of GlcNAc in a Modified E. coli Host

An E. coli mutant K-12 MG1655 strain optimized for GDP-fucoseproduction, as described in Example 1, was further mutated toadditionally produce GlcNAc. The mutations included a knock-out of theE. coli nagA and nagB genes together with a knock-in of constitutiveexpression constructs containing the mutant glmS*54 from E. colidiffering from the wild-type glmS protein (UniProt ID P17169) by anA39T, an R250C and an G4725 mutation and GNA1 from S. cerevisiae(UniProt ID P43577). The novel strain was evaluated and compared to itsparent strain in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contained 30 g/Lsucrose. Each strain was grown for 72 hours in multiple wells of a96-well plate. The experiment showed that 0.90±0.10 g/L GlcNAc could bemeasured in whole broth samples of the novel mutant strain whereas noGlcNAc production was detected in whole broth samples of the originalparent strain. The experiment further demonstrated that the mutationsadded for GlcNAc production did not affect the production of GDP-fucoseof the novel strain compared to the original parent strain.

Example 10: Production of GlcNAc and LacNAc in a Modified E. coli Host

An E. coli mutant K-12 MG1655 strain optimized for GDP-fucoseproduction, as described in Example 1, was further mutated with aknock-out of the E. coli nagA and nagB genes and with a genomic knock-inof a constitutive expression construct of the N-acetylglucosamineβ1,4-galactosyltransferase (LgtB) of N. meningitidis with SEQ ID NO:15.In a next step, cells of the mutant strain were transformed withdifferent constitutive expression vectors built up of distincttranscriptional units (TUs) containing the mutant glmS*54 from E. colidiffering from the wild-type glmS protein (UniProt ID P17169) by anA39T, an R250C and an G4725 mutation and GNA1 from S. cerevisiae(UniProt ID P43577) as enlisted in Tables 2 and 3. All novel strains(A-H) were evaluated in a growth experiment according to the cultureconditions provided in Example 1. Table 2 shows the production of GlcNAc(g/L) and LacNAc (g/L) in whole broth samples from each of the novelmutant E. coli strains, taken after 72 hours of cultivation in minimalmedium with 30 g/L sucrose. The data demonstrates that all new strainswere able to each produce both GlcNAc and LacNAc, without thesupplemental addition of GlcNAc to the culture medium. In contrast, areference strain sharing an identical genetic background but lacking anexpression plasmid for glmS*54 and GNA1 could only produce LacNAc inmedium supplemented with GlcNAc (Results not shown). Table 2 also showsthat the production titres of both GlcNAc and LacNAc in all novelstrains could be varied depending on the chosen transcriptional unitspresent in expression vectors A-H to express SEQ ID NOs: 15 and 19.

TABLE 2 Production of GlcNAc (g/L) and LacNAc (g/L) in whole brothsamples taken from mutant E. coli strains after 72 hours of cultivationin minimal medium comprising 30 g/L sucrose. TU for TU for GNA1 GlcNAcLacNAc glmS*54 (UniProt ID (g/L) (g/L) Strain (*) P43577) (±sd) (±sd) ATU45 TU44 0.98 (±0.07) 1.22 (±0.12) B TU53 TU44 1.90 (±0.14) 1.53(±0.13) C TU53 TU52 2.08 (±0.06) 1.45 (±0.08) D TU47 TU55 2.14 (±0.25)1.86 (±0.25) E TU46 TU44 2.40 (±0.22) 1.74 (±0.23) F TU45 TU57 2.81(±0.02) 1.60 (±0.08) G TU47 TU54 3.02 (±0.17) 2.19 (±0.17) H TU45 TU543.21 (±0.54) 2.26 (±0.46)

*differing from the wild-type glmS protein (UniProt ID P17169) by anA39T, an R250C and an G472S mutation

TABLE 3 Promoter and UTR sequences used in the transcriptional units(TUs) to express glmS*54 (differing from the wild-type glmS protein(UniProt ID P17169) by an A39T, an R250C and an G472S mutation) and GNA1(UniProt ID P43577) as shown in Table 2 TU Promoter* UTR* TerminatorTU44 Mutalik_P11 GalE_BCD18 rnpB_T1* TU45 Mutalik_P5 Gene10_LeuLT7early*** TU46 Mutalik_P5 GalE_LeuAB T7early*** TU47 Mutalik_P9Gene10_LeuL T7early*** TU52 Mutalik_P11 ThrA_BCD2 rnpB_T1** TU53Mutalik_P9 GalE_LeuAB T7early*** TU54 Mutalik_P10 GalE_BCD18 rnpB_T1**TU55 Mutalik_P10 GalE_BCD18 rnpB_T1** TU57 Mutalik_P10 ThrA_BCD2rnpB_T1** *Sequences taken from Mutalik et al. (Nat. Methods 2013, 10,354-360) **Sequences taken from Kim and Lee (FEBS letters 1997, 407(3),353-356) ***Sequences taken from Dunn et al. (Nucleic Acids Res. 1980,8(10), 2119-2132)

Example 11: Evaluation of LacNAc Production in a Modified E. coli Host

In a next experiment, an E. coli K-12 MG1655 strain optimized forGDP-fucose production and producing GlcNAc as described in Example 9,was further modified with a knock-in of the N-acetylglucosamineβ1,4-galactosyltransferase (LgtB) of N. meningitidis with SEQ ID NO:15.This novel strain together with a reference strain sharing the nagABknock-out and LgtB knock-in but expressing glmS*54 differing from thewild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and anG4725 mutation and GNA1 (UniProt ID P43577) from an identicaltranscription unit presented to the strain from plasmid were evaluatedin a growth experiment as described in Example 1. Table 4 shows theproduction of GlcNAc (g/L) and LacNAc (g/L) in whole broth samples fromeach of the mutant E. coli strains, taken after 72 hours of cultivationin minimal medium with 30 g/L sucrose. The data shows that both mutantstrains produce GlcNAc and LacNAc, independent of how both GNA1 andglmS*54 are presented to the strain.

TABLE 4 Production of GlcNAc (g/L) and LacNAc (g/L) in whole brothsamples taken from mutant E. coli strains after 72 hours of cultivationin minimal medium comprising 30 g/L sucrose Expression of atranscriptional GlcNAc (g/L) LacNAc unit for GNA1 (*) and glmS*54 (** )(±sd) (g/L) (±sd) From genomic knock-in 1.62 (±0.12) 1.60 (±0.06) Froman expression plasmid 6.71 (±0.57) 2.73 (±0.23) *UniProt ID P43577**differing from the wild-type glmS protein (UniProt ID P17169) by anA39T, an R250C and an G472S mutation

Example 12: Production of GlcNAc and LacNAc in Modified E. coli Hosts

In a next experiment, an E. coli K-12 MG1655 strain producing sialicacid, as described in Example 1 and containing knock-outs of the E. colinagA and nagB genes and genomic knock-ins of constitutive expressionconstructs containing glmS*54 differing from the wild-type glmS protein(UniProt ID P17169) by an A39T, an R250C and an G4725 mutation, the GNA1(UniProt ID P43577), the N-acetylglucosamine 2-epimerase (AGE) fromBacteroides ovatus (UniProt ID A7LVG6) and the N-acetylneuraminatesynthase (NeuB) from N. meningitidis (UniProt ID E0NCD4), was furthermodified with a knock-in of the N-acetylglucosamineβ1,4-galactosyltransferase (LgtB) of N. meningitidis with SEQ ID NO:15.

Also, an E. coli K-12 MG1655 strain optimized for enhanced UDP-galactoseproduction with genomic knock-outs of the E. coli ushA and galT genesand with a genomic knock-in of a constitutive expression construct forthe UDP-glucose-4-epimerase (galE) of E. coli (UniProt ID P09147) asdescribed in Example 1, was additionally mutated by a knock-out of theE. coli nagB gene and with a genomic knock-in of a constitutiveexpression construct of the gene encoding SEQ ID NO:15. The novelstrains were evaluated and compared to their respective parent strainsin a growth experiment according to the culture conditions provided inExample 1, in which the culture medium contained sucrose. Each strainwas grown for 72 hours in multiple wells of a 96-well plate. Theexperiment demonstrated that each novel strain produced GlcNAc andLacNAc whereas both parent strains did not produce LacNAc in whole brothsamples.

Example 13: Production of Galactosylated Oligosaccharides in a ModifiedE. coli Host

An E. coli K-12 MG1655 strain optimized for enhanced UDP-galactoseproduction and producing GlcNAc and LacNAc as described in Example 12,can additionally be transformed with an expression vector containing aconstitutive expression construct for the N-acetylglucosamineβ1,3-galactosyltransferase WbgO from E. coli O55:H7 with SEQ ID NO:03.The novel strain produces Gal-b14-(Galb13)-GlcNAc, containing twogalactose moieties linked beta-1,3 and beta-1,4 to GlcNAc, in additionto GlcNAc, LacNAc and LNB when evaluated in a growth experimentaccording to the culture conditions provided in Example 1, in which theculture medium contains sucrose.

Example 14: Production of Fucosylated LacNAc in a Modified E. coli Host

The E. coli K-12 MG1655 mutant strain H optimized for GDP-fucoseproduction and producing GlcNAc and LacNAc as described in Example 10,was additionally transformed with expression plasmids containing aconstitutive expression construct for either the a1,2-fucosyltransferaseHpFutC (GenBank NO. AAD29863.1) or the a1,3-fucosyltransferase HpFucT(UniProt ID O30511), both originating from Helicobacter pylori. Thenovel strains were evaluated in a growth experiment according to theculture conditions provided in Example 1. Table 5 shows the productionof 2′FLacNAc (g/L) or 3-FLacNAc (g/L) in whole broth samples from bothmutant strains, taken after 72 hours of cultivation in minimal mediumwith 30 g/L sucrose. The data demonstrates that each novel strainproduced GlcNAc, LacNAc and fucosylated forms of LacNAc, whereby2′FLacNAc was produced in the strain expressing HpFutC (GenBank NO.AAD29863.1) and 3-FLacNAc was produced in the strain expressing HpFucT(UniProt ID O30511). Difucosylated LacNAc was not detected in thisexperiment. The fucosylated LacNAc variants could not be detected inwhole broth samples of the parent strain of identical genetic backgroundlacking the expression vector for the fucosyltransferase.

TABLE 5 Production of 2′FLacNAc (g/L) and 3-FLacNAc (g/L) in whole brothsamples taken from mutant E. coli strains after 72 hours of cultivationin minimal medium comprising 30 g/L sucrose Mutant E. coli LacNAc2′FLacNAc 3-FLacNAc production strains expressing (g/L) (±sd) (g/L)(±sd) Reference (=no fucosyltransferase) 0 0 a1,2-fucosyltransferaseHpFutC 0.46 (±0.02) 0 (GenBank NO. AAD29863.1) a1,¾-fucosyltransferaseHpFucT 0 0.53 (±0.02) (UniProt ID O30511)

Example 15: Production of Di-Fucosylated LacNAc in a Modified E. coliHost

The E. coli K-12 MG1655 mutant strain H optimized for GDP-fucoseproduction and producing GlcNAc and LacNAc as described in Example 10,can additionally be transformed with an expression plasmid containingconstitutive expression constructs for both the fucosyltransferasesHpFutC (GenBank NO. AAD29863.1) and HpFucT (UniProt ID O30511). Thenovel strain produces 2′-fucosylated, 3-fucosylated and di-fucosylatedLacNAc in addition to GlcNAc and LacNAc in whole broth samples whenevaluated in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contains sucrose.

Example 16: Production of Sialylated LacNAc in a Modified E. coli HostCell

An E. coli K-12 MG1655 strain producing sialic acid as described inExample 1, was further mutated with a knock-in of a constitutiveexpression construct for LgtB with SEQ ID NO:15 and transformed with anexpression plasmid containing constitutive expression constructs for theN-acylneuraminate cytidylyltransferase (NeuA) from Pasteurella multocida(GenBank NO. AMK07891.1) and a PmultST3-like polypeptide consisting ofamino acid residues 1 to 268 of UniProt ID Q9CLP3 havingbeta-galactoside alpha-2,3-sialyltransferase activity. The novel strainwas evaluated and compared to its parent strain in a growth experimentaccording to the culture conditions provided in Example 1, in which theculture medium contained 30 g/L sucrose. Each strain was grown for 72hours in multiple wells of a 96-well plate. The data showed that thenovel strain produced 0.32±0.02 g/L a2,3-sialylated LacNAc in additionto GlcNAc and LacNAc in whole broth samples. Di-sialylated LacNAc couldnot be detected. The sialylated LacNAc variant could not be detected inwhole broth samples of the parent strain of identical genetic backgroundlacking the expression vector for the sialyltransferase.

Similarly, an E. coli K-12 MG1655 strain producing sialic acid asdescribed in Example 1, can further be mutated with a knock-in of aconstitutive expression construct for LgtB with SEQ ID NO:15 andtransformed with an expression plasmid containing constitutiveexpression constructs for NeuA from P. multocida (GenBank NO.AMK07891.1) and a PdST6-like polypeptide consisting of amino acidresidues 108 to 497 of UniProt ID O66375 having beta-galactosidealpha-2,6-sialyltransferase activity. The novel strain produces GlcNAcand LacNAc as well as a2,6-sialylated LacNAc in whole broth samples,when evaluated in a growth experiment according to the cultureconditions provided in Example 1, in which the culture medium containssucrose.

Example 17: Production of Di-Sialylated LacNAc in a Modified E. coliHost Cell

An E. coli K-12 MG1655 strain producing CMP-sialic acid as described inExample 1, can further be mutated with a knock-in of a constitutiveexpression construct for LgtB with SEQ ID NO:15 and transformed with anexpression plasmid containing constitutive expression constructs forboth the PmultST3-like polypeptide consisting of amino acid residues 1to 268 of UniProt ID Q9CLP3 having beta-galactosidealpha-2,3-sialyltransferase activity and the PdST6-like polypeptideconsisting of amino acid residues 108 to 497 of UniProt ID O66375 havingbeta-galactoside alpha-2,6-sialyltransferase activity. The novel strainproduces GlcNAc and LacNAc together with 3′-sialylated, 6′-sialylatedand di-sialylated LacNAc in whole broth samples when evaluated in agrowth experiment according to the culture conditions provided inExample 1, in which the culture medium contains sucrose.

Example 18: Production of Modified LacNAc in a E. coli Host

An E. coli K-12 MG1655 strain optimized for GDP-fucose production andproducing GlcNAc and LacNAc as described in Example 11, can additionallybe transformed with an expression plasmid containing a constitutiveexpression construct for the b1,3-N-acetyl-hexosaminyl-transferase lgtAfrom N. meningitidis (GenBank NO. AAM33849.1). By subsequent action ofthe mutant glmS*54 (differing from the wild-type glmS protein (UniProtID P17169) by an A39T, an R250C and an G472S mutation), the homologousEcGlmM and EcGlmU and the heterologous NmLgtA (GenBank NO. AAM33849.1),the thus created strain intracellularly converts fructose-6-phosphateinto UDP-GlcNAc, and uses this UDP-GlcNAc to intracellularly modifyLacNAc leading to production of GlcNAc-b1,3-Gal-b1,4-GlcNAc in wholebroth samples when evaluated in a growth experiment according to theculture conditions provided in Example 1, in which the culture mediumcontains sucrose. The novel strain also produces poly-LacNAc structures,i.e., (Gal-b1,4-GlcNAc)_(n) which are built of repeatedN-acetyllactosamine that are beta1,3-linked to each other by alternateactivity of LgtB with SEQ ID NO:15 and LgtA (GenBank NO. AAM33849.1)expressed in the strain.

An E. coli K-12 MG1655 strain optimized for UDP-galactose production andproducing GlcNAc and LacNAc as described in Example 12, was modified toconstitutively express the UDP-GlcNAc epimerase wbpP from Pseudomonasaeruginosa (UniProt ID Q8KN66) and the glycosyltransferase lgtD fromHaemophilus influenzae (UniProt ID A0A2X4DBP3). By subsequent action ofthe mutant E. coli glmS*54 (differing from the wild-type glmS protein(UniProt ID P17169) by an A39T, an R250C and a G4725 mutation), thehomologous E. coli glmM and glmU and the P. aeruginosa wbpP (UniProt IDQ8KN66), fructose-6-phosphate is intracellularly converted intoUDP-GalNAc via the intermediate compounds glucosamine-6-phoshpate,glucosamine-1-phosphate and UDP-GlcNAc. By subsequent action of thenewly expressed LgtD enzyme (UniProt ID A0A2X4DBP3), the novel strainwas able to modify the intracellularly produced LacNAc with GalNAc,producing 0.12±0.02 g/L GalNAc-b1,3-Gal-b1,4-GlcNAc in whole brothsamples when evaluated in a growth experiment according to the cultureconditions provided in Example 1, in which the culture medium contains30 g/L sucrose.

Example 19: Production of Sialylated Poly-LacNAc in a Modified E. coli

The E. coli strains producing LacNAc and sialylated forms of LacNAc inwhole broth samples as described in Example 16 and 17, can further betransformed with an expression plasmid containing a constitutiveexpression construct for LgtA (GenBank NO. AAM33849.1). By alternateactivity of the LgtB and LgtA transferases expressed with SEQ ID NO:15and GenBank NO. AAM33849.1, respectively, the novel strains additionallyproduce poly-LacNAc structures, i.e., (Gal-b1,4-GlcNAc)_(n) which arebuilt of repeated N-acetyllactosamine that are beta1,3-linked to eachother, together with sialylated poly-lacNAc structures in which Gal issialylated when evaluated in a growth experiment according to theculture conditions provided in Example 1. in which the culture mediumcontains sucrose.

Example 20: Production of LNB in a Modified E. coli Host

In a next experiment, an E. coli K-12 MG1655 strain optimized forGDP-fucose production and producing GlcNAc as described in Example 9,was further modified for constitutive expression of WbgO with SEQ IDNO:03, either from plasmid or from a genomic knock-in. The novel strainswere evaluated and compared to the parent strain lacking an expressionconstruct for SEQ ID NO:03 in a growth experiment according to theculture conditions provided in Example 1. Table 6 shows the productionof LNB (g/L) in whole broth samples from each of the mutant strains,taken after 72 hours of cultivation in minimal medium with 30 g/Lsucrose. The data demonstrates that both novel strains produced LNB inwhole broth samples, independent of how the WbgO was presented to thestrain.

TABLE 6 Production of LNB (g/L) in whole broth samples taken from mutantE. coli strains after 72 hours of cultivation in minimal mediumcomprising 30 g/L sucrose Expression of a transcriptional unit LNB forEc WbgO with SEQ ID NO: 03 (g/L) (±sd) From genomic knock-in 0.63(±0.12) From an expression plasmid 2.81 (±0.11)

Example 21: Production of Fucosylated LNB Forms in a Modified E. coliHost

An E. coli K-12 MG1655 strain optimized for GDP-fucose production andproducing GlcNAc and LNB as described in Example 20 wherein WbgO withSEQ ID NO:03 was expressed from a genomic knock-in, was additionallytransformed with expression plasmids containing a constitutiveexpression construct for either the a1,2-fucosyltransferase HpFutC(GenBank NO. AAD29863.1) or the a1,3-fucosyltransferase HpFucT (UniProtID O30511), both originating from Helicobacter pylori. The novel strainswere evaluated in a growth experiment according to the cultureconditions provided in Example 1. Table 7 shows the production of 2′FLNB(g/L) or 4-FLNB (g/L) in whole broth samples from both mutant strains,taken after 72 hours of cultivation in minimal medium with 30 g/Lsucrose. The data demonstrates that the novel strains each producedfucosylated forms of LNB in whole broth samples whereby 2′FLNB wasmeasured in the strain expressing HpFutC (GenBank NO. AAD29863.1) and4-FLNB was measured in the strain expressing HpFucT (UniProt ID O30511).Di-fucosylated LNB was not detected in the experiment. The fucosylatedLNB variants could not be detected in whole broth samples of the parentstrain of identical genetic background lacking the expression vector forthe fucosyltransferase.

TABLE 7 Production of 2′'FLNB (g/L) and 4-FLNB (g/L) in whole brothsamples taken from mutant E. coli strains after 72 hours of cultivationin minimal medium comprising 30 g/L sucrose Mutant E. coli LNB 2′FLNB(g/L) 4-FLNB (g/L) production strains expressing (±sd) (±sd) Reference(=no fucosyltransferase) 0 0 a1,2-fucosyltransferase HpFutC 0.19 (±0.03)0 (GenBank NO. AAD29863.1) a1,3-fucosyltransferase HpFucT 0 0.04 (±0.01)(UniProt ID O30511)

Example 22: Production of Difucosylated LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain optimized for GDP-fucose production andproducing GlcNAc and LNB, as described in Example 20 wherein WbgO withSEQ ID NO:03 is expressed from a genomic knock-in, can additionally betransformed with an expression plasmid containing constitutiveexpression constructs for both the a1,2-fucosyltransferase HpFutC(GenBank NO. AAD29863.1) and the a1,3-fucosyltransferase HpFucT (UniProtID O30511). The novel strain produces GlcNAc and LNB together with2′-fucosylated, 4-fucosylated and di-fucosylated LNB in whole brothsamples when evaluated in a growth experiment according to the cultureconditions provided in Example 1, in which the culture medium containssucrose.

Example 23: Production of Galactosylated LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain optimized for enhanced production ofUDP-galactose as described in Example 1, was additionally mutated by aknock-out of the E. coli nagB gene and was further modified forconstitutive expression of WbgO with SEQ ID NO:03, either from plasmidor from a genomic knock-in. Both novel strains produce LNB in wholebroth samples when evaluated in a growth experiment according to theculture conditions provided in Example 1, in which the culture mediumcontains sucrose. When the novel LNB production strains are additionallytransformed with expression constructs for analpha-1,2-galactosyltransferase and/or alpha-1,3-galactosyltransferaseand/or beta-1,3-galactosyltransferase and/orbeta-1,4-galactosyltransferase each of the novel strains produces GlcNAcand LNB together with galactosylated LNB forms in whole broth samples,when evaluated in a growth experiment according to the cultureconditions provided in Example 1, in which the culture medium containssucrose.

Example 24: Production of Sialylated LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain producing CMP-sialic acid, as described inExample 1, can further be mutated with a knock-in of a constitutiveexpression construct for WbgO with SEQ ID NO:03 and transformed with anexpression plasmid containing a constitutive expression construct for aPmultST3-like polypeptide consisting of amino acid residues 1 to 268 ofUniProt ID Q9CLP3 having beta-galactoside alpha-2,3-sialyltransferaseactivity. The novel strain produces in addition to GlcNAc and LNB alsosialylated LNB forms in whole broth samples, when evaluated in a growthexperiment according to the culture conditions provided in Example 1, inwhich the culture medium contains sucrose. Similarly, an E. coli K-12MG1655 CMP-sialic production strain additionally expressing WbgO withSEQ ID NO:03 and a PdST6-like polypeptide consisting of amino acidresidues 108 to 497 of UniProt ID O66375 having beta-galactosidealpha-2,6-sialyltransferase activity produces GlcNAc and LNB togetherwith sialylated LNB forms in whole broth samples, when evaluated in agrowth experiment according to the culture conditions provided inExample 1, in which the culture medium contains sucrose.

Example 25: Production of Di-Sialylated LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain producing CMP-sialic acid, as described inExample 1, can further be mutated with a knock-in of a constitutiveexpression construct for WbgO with SEQ ID NO:03 and transformed with anexpression plasmid containing (a) constitutive expression construct(s)for a PmultST3-like polypeptide consisting of amino acid residues 1 to268 of UniProt ID Q9CLP3 having beta-galactosidealpha-2,3-sialyltransferase activity and/or a PdST6-like polypeptideconsisting of amino acid residues 108 to 497 of UniProt ID O66375 havingbeta-galactoside alpha-2,6-sialyltransferase activity. The novel strainproduces GlcNAc and LNB together with mono-sialylated and/ordi-sialylated LNB in whole broth samples, when evaluated in a growthexperiment according to the culture conditions provided in Example 1, inwhich the culture medium contains sucrose.

Example 26: Fermentative Production of LacNAc with a Mutant E. coli Host

A mutant E. coli K-12 MG1655 strain, optimized for GDP-fucose productionand producing GlcNAc and LacNAc with glmS*54 (differing from thewild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and anG472S mutation) and GNA1 (UniProt ID P43577) presented to the host fromexpression plasmid as described in Example 11, was evaluated in afed-batch fermentation at 5 L bioreactor scale according to theconditions provided in Example 1. In this example, sucrose was used as acarbon source. Regular samples were taken and the production of LacNAcwas measured, as described in Example 1.

Example 27: Production of LacNAc or LNB in a Modified E. coli Host whenGrown on Other Carbon Sources than Sucrose

Mutant E. coli strains modified for the production of GlcNAc and LacNAcas described in Examples 10 to 12 or mutant E. coli strains modified forthe production of GlcNAc and LNB as described in Example 20 produceGlcNAc and LacNAc or GlcNAc and LNB, respectively, when evaluated in agrowth experiment according to the culture conditions provided inExample 1, in which the culture medium contains glycerol. The mutantstrains also produce GlcNAc and LacNAc or GlcNAc and LNB, respectively,when evaluated in fed-batch fermentations at bioreactor scale, asdescribed in Example 1, using any one or more of but not limited tofollowing carbon sources: glycerol, glucose, fructose, lactose,arabinose, maltotriose, sorbitol, xylose, rhamnose and mannose.

Example 28: Materials and Methods in Saccharomyces cerevisiae

Media

Strains are grown on Synthetic Defined yeast medium with CompleteSupplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura) containing 6.7g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/Lagar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/Llactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura (MP Biomedicals).

Strains

Saccharomyces cerevisiae BY4742 created by Brachmann et al. (Yeast(1998) 14:115-32) was used, available in the Euroscarf culturecollection. All mutant strains were created by homologous recombinationor plasmid transformation using the method of Gietz (Yeast 11:355-360,1995).

Plasmids

Yeast expression plasmid p2a_2μ (Chan 2013, Plasmid 70, 2-17) was usedfor expression of foreign genes in Saccharomyces cerevisiae. Thisplasmid contained an ampicillin resistance gene and a bacterial originof replication to allow for selection and maintenance in E. coli. Theplasmid further contained the 2μ yeast ori and the Ura3 selection markerfor selection and maintenance in yeast.

In one example, the yeast expression plasmid p2a_2μ can be modified toobtain the mutant fructose-6-phosphate aminotransferase glmS*54 from E.coli (differing from the wild-type glmS protein (UniProt ID P17169) byan A39T, an R250C and an G472S mutation) as described in WO 2018122225,the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae(UniProt ID P43577), a phosphatase like any one or more of e.g., the E.coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP,YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV,YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonasputida, ScDOG1 from S. cerevisiae or BsAraL from Bacillus subtilis asdescribed in WO 2018122225. The modified plasmids can further bemodified to obtain an N-acetylglucosamine b-1,3-galactosyltransferasechosen from the list comprising SEQ ID NOs: 03, 04, 05, 06, 07, 08, 10,11, 12 and 13 and/or an N-acetylglucosamine b-1,4-galactosyltransferasechosen from the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21,22, 23, 26, 27, 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41.

In one example to produce GDP-fucose, a yeast expression plasmid likep2a_2μ_Fuc (Chan 2013, Plasmid 70, 2-17) d is further modified withconstitutive transcriptional units for a lactose permease like e.g.,LAC12 from K lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydrataselike e.g., gmd from E. coli (UniProt ID POAC88) and a GDP-L-fucosesynthase like e.g., fcl from E. coli (UniProt ID P32055). The yeastexpression plasmid p2a_2μ_Fuc2 can be used as an alternative expressionplasmid of the p2a_2μ_Fuc plasmid comprising next to the ampicillinresistance gene, the bacterial ori, the 2μ yeast ori and the Ura3selection marker constitutive transcriptional units for a lactosepermease like e.g., LAC12 from K. lactis (UniProt ID P07921), a fucosepermease like e.g., fucP from E. coli (UniProt ID P11551) and abifunctional enzyme with fucose kinase/fucose-1-phosphateguanylyltransferase activity like e.g., fkp from Bacteroides fragilis(UniProt ID SUV40286.1). To further produce fucosylatedoligosaccharides, the p2a_2μ_Fuc and its variant the p2a_2μ_Fuc2,additionally contained a constitutive transcriptional unit for analpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori (GenBankNo. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like e.g., HpFucTfrom H. pylori (UniProt ID O30511).

In one example to produce UDP-galactose, a yeast expression plasmid canbe derived from the pRS420-plasmid series (Christianson et al., 1992,Gene 110: 119-122) containing the HIS3 selection marker and aconstitutive transcriptional unit for an UDP-glucose-4-epimerase likee.g., galE from E. coli (UniProt ID P09147). This plasmid can be furthermodified with constitutive transcriptional units for a lactose permeaselike e.g., LAC12 from K. lactis (UniProt ID P07921) and a galactosidebeta-1,3-N-acetylglucosaminyltransferase activity like e.g., lgtA fromN. meningitidis (UniProt ID Q9JXQ6) to produce LN3. To further produceLN3-derived oligosaccharides like LNT, the mutant LN3 producing strainswere further modified with a constitutive transcriptional unit for anN-acetylglucosamine beta-1,3-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 03, 04, 05, 06, 07, 08, 10, 11, 12 and 13. Tofurther produce LN3-derived oligosaccharides like LNnT, the mutant LN3producing strains were further modified with a constitutivetranscriptional unit for an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40 and 41.

In one example to produce sialic acid and CMP-sialic acid, a yeastexpression plasmid can be derived from the pRS420-plasmid series(Christianson et al., 1992, Gene 110: 119-122) containing the TRP1selection marker and constitutive transcriptional units for one or morecopies of an L-glutamine-D-fructose-6-phosphate aminotransferase likee.g., the mutant glmS*54 from E. coli (differing from the wild-type E.coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G4725mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), onephosphatase like any one or more of e.g., the E. coli genes comprisingaphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX,YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL,YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S.cerevisiae or BsAraL from Bacillus subtilis as described in WO2018122225, an N-acetylglucosamine 2-epimerase like e.g., AGE from B.ovatus (UniProt ID A7LVG6), an N-acetylneuraminate synthase like e.g.,from Neisseria meningitidis (UniProt ID E0NCD4), and anN-acylneuraminate cytidylyltransferase like e.g., the NeuA enzyme fromC. jejuni (UniProt ID Q93MP7), the NeuA enzyme from Haemophilusinfluenzae (GenBank No. AGV11798.1) or the NeuA enzyme from Pasteurellamultocida (GenBank No. AMK07891.1). Optionally, a constitutivetranscriptional unit comprising one or more copies for a glucosamine6-phosphate N-acetyltransferase like e.g., GNA1 from S. cerevisiae(UniProt ID P43577) was/were added as well. To produce sialylatedoligosaccharides, the plasmid further comprised constitutivetranscriptional units for a lactose permease like e.g., LAC12 fromKluyveromyces lactis (UniProt ID P07921), and one or more copies of abeta-galactoside alpha-2,3-sialyltransferase like e.g., PmultST3 from P.multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consistingof amino acid residues 1 to 268 of UniProt ID Q9CLP3 havingbeta-galactoside alpha-2,3-sialyltransferase activity, NmeniST3 from N.meningitidis (GenBank No. ARC07984.1) or PmultST2 from P. multocidasubsp. multocida str. Pm70 (GenBank No. AAK02592.1), a beta-galactosidealpha-2,6-sialyltransferase like e.g., PdST6 from Photobacteriumdamselae (UniProt ID O66375) or a PdST6-like polypeptide consisting ofamino acid residues 108 to 497 of UniProt ID O66375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt IDQ64689).

Preferably, but not necessarily, the glycosyltransferase proteins and/orthe proteins involved in nucleotide-activated sugar synthesis wereN-terminally fused to a SUMOstar tag (e.g., obtained from pYSUMOstar,Life Sensors, Malvern, PA) to enhance their solubility.

Optionally, the mutant yeast strains were modified with a knock-ins of aconstitutive transcriptional unit encoding a chaperone protein likee.g., Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2,Ssa3, Ssa4, Ssb2, Ecm10, Ssc1, Ssq1, Ssz1, Lhs1, Hsp82, Hsc82, Hsp78,Hsp104, Tcp1, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6, or Cct7 (Gong et al.,2009, Mol. Syst. Biol. 5: 275).

Plasmids were maintained in the host E. coli DH5alpha (F⁻,phi80d/acZdeltaM15, delta(lacZYA-argF)U169, deoR, recA1, endA1,hsdR17(rk⁻, mk⁺), phoA, supE44, lambda⁻, thi-1, gyrA96, relA1) boughtfrom Invitrogen.

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from thegenome were synthetically synthetized with one of the followingcompanies: DNA2.0, Gen9, IDT or Twist Bioscience.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivations Conditions

In general, yeast strains were initially grown on SD CSM plates toobtain single colonies. These plates were grown for 2-3 days at 30° C.

Starting from a single colony, a preculture was grown over night in 5 mLat 30° C., shaking at 200 rpm. Subsequent 125 mL shake flask experimentswere inoculated with 2% of this preculture, in 25 mL media. These shakeflasks were incubated at 30° C. with an orbital shaking of 200 rpm.

Gene Expression Promoters

Genes were expressed using synthetic constitutive promoters, asdescribed by Blazeck (Biotechnology and Bioengineering, Vol. 109, No.11, 2012).

Example 29: Production of GlcNAc and LacNAc; or GlcNAc and LNB in S.cerevisiae

Another example provides use of an eukaryotic organism, in the form ofSaccharomyces cerevisiae, for performing the disclosure. Using thestrains, plasmids and methods as described in Example 28, a mutant S.cerevisiae strain is created that produces GlcNAc and LacNAc. Thesemodifications comprise the addition of constitutive expression units forthe mutant fructose-6-phosphate aminotransferase glmS*54 of E. coli(differing from the wild-type E. coli glmS, having UniProt ID P17169, byan A39T, an R250C and an G4725 mutation), the glucosamine 6-phosphateN-acetyltransferase GNA1 of S. cerevisiae (UniProt ID P43577), onephosphatase chosen from the list comprising any one or more of the E.coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP,YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV,YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonasputida, ScDOG1 from S. cerevisiae and BsAraL from Bacillus subtilis asdescribed in WO 2018122225 and the N-acetylglucosamineb-1,4-galactosyltransferase LgtB of N. meningitidis of SEQ ID NO:15. Themutant S. cerevisiae strain is capable of growing on glucose or glycerolas carbon source, converting the carbon source intofructose-6-phosphate, which is then converted to GlcNAc and subsequentlyLacNAc, by activity of the novel expressed fructose-6-phosphateaminotransferase, glucosamine 6-phosphate N-acetyltransferase, thephosphatase and N-acetylglucosamine b-1,4-galactosyltransferase.

Preculture of the strain is made in 5 mL of the synthetic defined mediumSD-CSM containing 22 g/L glucose and grown at 30° C. as described inExample 28. This preculture is then inoculated in 25 mL medium in ashake flask with 10 g/L glucose as sole carbon source and grown at 30°C. Regular samples are taken and the production of GlcNAc and LacNAc ismeasured as described in Example 1. A similar yeast strain containingthe N-acetylglucosamine b-1,3-galactosyltransferase WbgO of E. coliO55:H7 with SEQ ID NO:03 instead of the N-acetylglucosamineb-1,4-galactosyltransferase LgtB of SEQ ID NO:15 is capable of producingGlcNAc and LNB in a similar cultivation experiment.

Example 30: RegEx Search for N-Acetylglucosamineb-1,3-Galactosyltransferase Genes Having PFAM Domain PF00535

A RegEx analysis was performed for the N-acetylglucosamineb-1,3-galactosyltransferase genes having PFAM domain PF00535 to findmembers comprising the sequence [AGPS]XXLN(X_(n))RXDXD with SEQ IDNO:01, wherein X is any amino acid and wherein n is 12 to 17, or to findmembers comprising the sequencePXXLN(X_(n))RXDXD(X_(m))[FWY]XX[HKR]XX[NQST] with SEQ ID NO:02, whereinX is any amino acid and wherein n is 12 to 17 and m 100 to 115. To thisend, all N-acetylglucosamine b-1,3-galactosyltransferase genes havingPFAM domain PF00535 as annotated in the Pfam database version Pfam 33.1(as released on 11 Jun. 2020) were downloaded from the UniProt database(as released on 3 Jul. 2020) and analyzed for the presence of the motifsaccording the method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on 6 Apr. 2019). Corresponding members from the RegExsearch comprised A0A3545D93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6,A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0,A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6,A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2,A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63,A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3.

Example 31: RegEx search for other N-acetylglucosamineb-1,3-galactosyltransferase or N-acetylglucosamineb-1,4-galactosyltransferase genes

A RegEx analysis can be performed for the N-acetylglucosamineb-1,3-galactosyltransferase genes having PFAM domain domain IPR002659 tofind members comprising the sequenceKT(X_(n))[FY]XXKXDXD(X_(m))[FHY]XXG(X, no A, G, S)(X_(p))X(no F, H, W,Y)[DE]D[ILV]XX[AG] with SEQ ID NO:09, wherein X is any amino acid andwherein n is 13 to 16, m 35 to 70 and p 20 to 45. To this end, allN-acetylglucosamine b-1,3-galactosyltransferase genes having PFAM domainIPR002659 as annotated in the Pfam database version Pfam 33.1 (asreleased on 11 Jun. 2020) were downloaded from the UniProt database (asreleased on 3 Jul. 2020) and analyzed for the presence of the motifsaccording the method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on 6 Apr. 2019).

A similar RegEx analysis can be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF01755 to findmembers comprising the sequenceEXXCXXSHXX[ILV][FWY](X_(n))EDD(X_(m))[ACGST]XXYX[ILMV] with SEQ IDNO:14, wherein X is any amino acid and wherein n is 13 to 15 and m 50 to76. To this end, all N-acetylglucosamine b-1,4-galactosyltransferasegenes having PFAM domain PF01755 as annotated in the Pfam databaseversion Pfam 33.1 (as released on 11 Jun. 2020) were downloaded from theUniProt database (as released on 3 Jul. 2020) and analyzed for thepresence of the motifs according the method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on 6 Apr. 2019).

Similarly, a RegEx analysis can be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF00535 to findmembers comprising the sequenceR[KN]XXXXXXXGXXXX[FL]XDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE] with SEQ IDNO:24, wherein X is any amino acid and wherein n is 50 to 75 and m 10 to30, or members comprising the sequenceR[KN]XXXXXXXGXXXXFXDXD(X_(n))[FHW]XXX[FHNY](X_(m))E[DE](X_(p))[FWY]XX[HKR]XX[NQST]with SEQ ID NO:25, wherein X is any amino acid and wherein n is 50 to75, m is 10 to 30 and p is 20 to 25. To this end, allN-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domainPF00535 as annotated in the Pfam database version Pfam 33.1 (as releasedon 11 Jun. 2020) were downloaded from the UniProt database (as releasedon 3 Jul. 2020) and analysed for the presence of the motifs accordingthe method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on 6 Apr. 2019).

A similar RegEx analysis can be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF02709 and nothaving PFAM domain PF00535 to find members comprising the sequence[FWY]XX[FY][FWY](X₂₃)[FWY][GQ]X[DE]D with SEQ ID NO:29, wherein X is anyamino acid, or members comprising the sequence[PV]W[GHNP](X_(n))[FWY][GQ]X[DE]D with SEQ ID NO:30, wherein X is anyamino acid and wherein n is 21 to 24. To this end, allN-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domainPF02709 and not having PFAM domain PF00535 as annotated in the Pfamdatabase version Pfam 33.1 (as released on 11 Jun. 2020) were downloadedfrom the UniProt database (as released on 3 Jul. 2020) and analysed forthe presence of the motifs according the method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2 (as released on 6 Apr. 2019).

Finally, a RegEx analysis can be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF03808 to findmembers comprising the sequence[ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA with SEQ ID NO:37, whereinX is any amino acid and wherein n is 20 to 25, or members comprising thesequence[ST][FHY]XN(X_(n))DG(X₁₆)[HKR]X[ST]FDXX[ST]XA(X_(m))[HR]XG[FWY](X_(p))GXGXXXQ[DE] with SEQ ID NO:38, wherein X is any amino acid and wherein n is 20 to25, m is 40 to 50 and p is 22 to 30. To this end, allN-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domainPF03808 as annotated in the Pfam database version Pfam 33.1 (as releasedon 11 Jun. 2020) were downloaded from the UniProt database (as releasedon 3 Jul. 2020) and analysed for the presence of the motifs accordingthe method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on 6 Apr. 2019).

Example 32: Production of an Oligosaccharide Mixture Comprising 6′-SL,LacNAc, Sialylated LacNAc, LN3, Sialylated LN3, LNnT and LSTc with aModified E. coli Host

An E. coli K-12 strain MG1655 is modified for sialic acid production asdescribed in Example 1 comprising knock-outs of the E. coli nagA, nagB,nanA, nanT, nanE, nanK, LacZ, LacY and LacA genes and genomic knock-insof constitutive transcriptional units comprising genes encoding thelactose permease (LacY) from E. coli (UniProt ID P02920), the sialicacid transporter (nanT) from E. coli (UniProt ID P41036), the mutantL-glutamine-D-fructose-6-phosphate aminotransferase glmS*54 from E. coli(differing from the wild-type E. coli glmS, having UniProt ID P17169, byan A39T, an R250C and an G472S mutation), the glucosamine 6-phosphateN-acetyltransferase (GNA1) from S. cerevisiae (UniProt ID P43577), theN-acetylglucosamine 2-epimerase (AGE) from B. ovatus (UniProt IDA7LVG6), the N-acetylneuraminate synthase (NeuB) from C. jejuni (UniProtID Q931MP9), the sucrose transporter (CscB) from E. coli W (UniProt IDE0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417)and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt IDA0ZZH6). The thus obtained mutant E. coli strain producing sialic acidis further modified with a genomic knock-in of constitutivetranscriptional units to express the N-acylneuraminatecytidylyltransferase enzyme NeuA from C. jejuni (UniProt ID Q93MP7) andthe alpha-2,6-sialyltransferase PdbST from P. damselae (UniProt IDO66375) to produce 6′-sialyllactose. In a next step, the mutant strainis further modified with genomic knock-ins comprising constitutivetranscriptional units for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(UniProt ID Q9JXQ6) and an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40 and 41 to produce a mixture of oligosaccharides comprising6′-SL, LacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and LSTc(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc). The novel strains areevaluated in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contains sucrose andlactose. The strains are grown in four biological replicates in a96-well plate. After 72h of incubation, the culture broth is harvested,and the sugars are analysed on UPLC.

Example 33: Production of an Oligosaccharide Mixture LN3, SialylatedLN3, LNT, LNB, Sialylated LNB, 3′-SL and LSTa with a Modified E. coliHost

An E. coli strain modified to produce sialic acid (Neu5Ac) as describedin Example 32 is further modified with a genomic knock-in ofconstitutive transcriptional units to express the N-acylneuraminatecytidylyltransferase enzyme NeuA from C. jejuni (UniProt ID Q93MP7) andthe alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt IDQ9CLP3) to produce 3′-siayllactose. In a next step, the mutant strain isfurther modified with genomic knock-ins comprising constitutivetranscriptional units for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(UniProt ID Q9JXQ6) and an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13 to produce a mixture ofoligosaccharides comprising LN3, 3′-sialylated LN3(Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc), LNT, LNB, sialylated LNB, 3′-SLand LSTa (Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b-Gal-b1,4-Glc). The novel strainsare evaluated in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contains sucrose andlactose. The strains are grown in four biological replicates in a96-well plate. After 72 h of incubation, the culture broth is harvested,and the sugars are analysed on UPLC.

Example 34: Material and Methods Bacillus subtilis

Media

Two different media are used, namely a rich Luria Broth (LB) and aminimal medium for shake flask (MMsf). The minimal medium uses a traceelement mix.

Trace element mix consisted of 0.735 g/L CaCl₂·2H₂O, 0.1 g/L MnCl₂·2H₂O,0.033 g/L CuCl₂·2H₂O, 0.06 g/L CoCl₂·6H₂O, 0.17 g/L ZnCl₂, 0.0311 g/LH₃BO₄, 0.4 g/L Na₂EDTA·2H₂O and 0.06 g/L Na₂MoO₄. The Fe-citratesolution contained 0.135 g/L FeCl₃·6H₂O, 1 g/L Na-citrate (Hoch 1973PMC1212887).

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco,Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodiumchloride (VWR. Leuven, Belgium). Luria Broth agar (LBA) plates consistedof the LB media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

The minimal medium for the shake flasks (MMsf) experiments contained2.00 g/L (NH₄)₂SO₄, 7.5 g/L KH₂PO₄, 17.5 g/L K2HPO₄, 1.25 g/LNa-citrate, 0.25 g/L MgSO₄·7H₂O, 0.05 g/L tryptophan, from 10 up to 30g/L glucose or another carbon source including but not limited tofructose, maltose, sucrose, glycerol and maltotriose when specified inthe examples, 10 ml/L trace element mix and 10 ml/L Fe-citrate solution.The medium was set to a pH of 7 with 1M KOH. Depending on the experimentsialic acid or lactose could be added as a precursor.

Complex medium, e.g., LB, was sterilized by autoclaving (121° C., 21′)and minimal medium by filtration (0.22 μm Sartorius). When necessary,the medium was made selective by adding an antibiotic (e.g., zeocin (20mg/L)).

Strains, Plasmids and Mutations

Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio,USA).

Plasmids for gene deletion via Cre/lox are constructed as described byYan et al. (Appl. & Environm. Microbial., September 2008, p5556-5562).Gene disruption is done via homologous recombination with linear DNA andtransformation via electroporation as described by Xue et al. (J.Microb. Meth. 34 (1999) 183-191). The method of gene knockouts isdescribed by Liu et al. (Metab. Engine. 24 (2014) 61-69). This methoduses 1000 bp homologies up- and downstream of the target gene.

Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7,15158) are used as expression vector and could be further used forgenomic integrations if necessary. A suitable promoter for expressioncan be derived from the part repository (iGem): sequence id:Bba_K143012, Bba_K823000, Bba_K823002 or Bba_K823003. Cloning can beperformed using Gibson Assembly, Golden Gate assembly, Cliva assembly,LCR or restriction ligation.

In an example for the production of LNB, Bacillus subtilis mutantstrains are modified with genomic knock-ins comprising constitutivetranscriptional units for the mutant glmS*54 from E. coli (differingfrom the wild-type E. coli glmS, having UniProt ID P17169, by an A39T,an R250C and an G4725 mutation as described by Deng et al. (Biochimie88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from thelist comprising any one or more of the E. coli genes comprising aphA,Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC,YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG,YrfG and YbiU or PsMupP from Pseudomonas putida, DOG1 from S. cerevisiaeor AraL from Bacillus subtilis as described in WO 2018122225 and anN-acetylglucosamine beta-1,3-galactosyltransferase chosen from the listcomprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13.

In an example for the production of LacNAc, Bacillus subtilis mutantstrains are modified with genomic knock-ins comprising constitutivetranscriptional units for the mutant glmS*54 from E. coli (differingfrom the wild-type E. coli glmS, having UniProt ID P17169, by an A39T,an R250C and an G4725 mutation as described by Deng et al. (Biochimie88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from thelist comprising any one or more of the E. coli genes comprising aphA,Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC,YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG,YrfG and YbiU or PsMupP from Pseudomonas putida, DOG1 from S. cerevisiaeor AraL from Bacillus subtilis as described in WO 2018122225 and anN-acetylglucosamine beta-1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28,31, 32, 33, 34, 35, 36, 39, 40 and 41. To further fucosylate the LNB orLacNAc, the LNB or LacNAc producing strains are further modified with aconstitutive transcriptional unit for an alpha-1,2-fucosyltransferaselike e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or analpha-1,3-fucosyltransferase like e.g., HpFucT from H. pylori (UniProtID O30511).

In an example for the production of lactose-based oligosaccharides,Bacillus subtilis mutant strains are created to contain a gene codingfor a lactose importer (such as e.g., the E. coli LacY with UniProt IDP02920). For 2′FL, 3FL and diFL production, an alpha-1,2- and/oralpha-1,3-fucosyltransferase expression construct is additionally addedto the strains.

In an example for the production of lacto-N-triose (LNT-II, LN3,GlcNAc-b1,3-Gal-b1,4-Glc), the B. subtilis strain is modified with agenomic knock-in of constitutive transcriptional units comprising alactose importer (such as e.g., the E. coli LacY with UniProt ID P02920)and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g.,LgtA from N. meningitidis (GenBank: AAM33849.1). For LNT production, theLN3 producing strain is further modified with a constitutivetranscriptional unit for an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13, that can be delivered tothe strain either via genomic knock-in or from an expression plasmid.For LNnT production, the LN3 producing strain is further modified with aconstitutive transcriptional unit for an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40 and 41. To further fucosylate the LN3, LNT or LNnT, themutant strains are further modified with a constitutive transcriptionalunit for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H.pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferaselike e.g., HpFucT from H. pylori (UniProt ID O30511).

In an example for sialic acid production, a mutant B. subtilis strain iscreated by overexpressing the native fructose-6-P-aminotransferase(UniProt ID POCI73) to enhance the intracellular glucosamine-6-phosphatepool. Further on, the enzymatic activities of the genes nagA, nagB andgamA are disrupted by genetic knockouts and aglucosamine-6-P-aminotransferase from S. cerevisiae (UniProt ID P43577),an N-acetylglucosamine-2-epimerase from B. ovatus (UniProt ID A7LVG6)and an N-acetylneuraminate synthase from C. jejuni (UniProt ID Q93MP9)are overexpressed on the genome. To allow sialylated oligosaccharideproduction, the sialic acid producing strain is further modified withexpression constructs comprising an N-acylneuraminatecytidylyltransferase like e.g., the NeuA enzyme from C. jejuni (UniProtID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV11798.1)or the NeuA enzyme from P. multocida (GenBank No. AMK07891.1), and oneor more copies of a beta-galactoside alpha-2,3-sialyltransferase likee.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-likepolypeptide consisting of amino acid residues 1 to 268 of UniProt IDQ9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity,NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 fromP. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), abeta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 fromPhotobacterium damselae (UniProt ID O66375) or a PdST6-like polypeptideconsisting of amino acid residues 108 to 497 of UniProt ID O66375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt IDQ64689). In an example for the production of lactose-based sialylatedoligosaccharides, the Bacillus subtilis mutant strains are furthermodified with a constitutive transcriptional unit for a lactose importer(such as e.g., the E. coli LacY with UniProt ID P02920).

For growth on sucrose, the mutant strains can additionally be modifiedwith genomic knock-ins of constitutive transcriptional units comprisingthe sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), thefructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and thesucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from thegenome were synthetically synthetized with one of the followingcompanies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivation Conditions

A preculture of 96-well microtiter plate experiments was started from acryovial or a single colony from an LB plate, in 150 μL LB and wasincubated overnight at 37° C. on an orbital shaker at 800 rpm. Thisculture was used as inoculum for a 96-well square microtiter plate, with400 μL MMsf medium by diluting 400x. Each strain was grown in multiplewells of the 96-well plate as biological replicates. These final 96-wellculture plates were then incubated at 37° C. on an orbital shaker at 800rpm for 72 h, or shorter, or longer. At the end of the cultivationexperiment samples were taken from each well to measure the supernatantconcentration (extracellular sugar concentrations, after 5 min. spinningdown the cells), or by boiling the culture broth for 15 min at 90° C. orfor 60 min at 60° C. before spinning down the cells (=whole brothconcentration, intra- and extracellular sugar concentrations, as definedherein).

Also, a dilution of the cultures was made to measure the optical densityat 600 nm. The cell performance index or CPI was determined by dividingthe oligosaccharide concentrations by the biomass, in relativepercentages compared to a reference strain. The biomass is empiricallydetermined to be approximately ⅓rd of the optical density measured at600 nm.

Example 35: Production of 2′FLNB with a Modified B. subtilis Strain

A B. subtilis strain is first modified for LNB production and growth onsucrose by genomic knock-out of the nagB, glmS and gamA genes andgenomic knock-ins of constitutive transcriptional units comprising genesencoding the native fructose-6-P-aminotransferase (UniProt ID P0CI73),the mutant glmS*54 from E. coli (differing from the wild-type E. coliglmS, having UniProt ID P17169, by an A39T, an R250C and an G472Smutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), theglucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae(UniProt ID P43577), the phosphatase AraL from B. subtilis (UniProt IDP94526), an N-acetylglucosamine beta-1,3-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 03, 04, 05, 06, 07, 08, 10, 11, 12or 13, the sucrose transporter (CscB) from E. coli W (UniProt IDE0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417)and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt IDA0ZZH6). In a next step, the LNB producing strain is transformed with anexpression plasmid comprising a constitutive transcriptional unit forthe alpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori(GenBank No. AAD29863.1).

The novel strain is evaluated for the production 2′FLNB in a growthexperiment on MMsf medium lacking a precursor according to the cultureconditions provided in Example 34. After 72 h of incubation, the culturebroth is harvested, and the sugars are analysed on UPLC.

Example 36: Production of an Oligosaccharide Mixture Comprising 6′-SL,LacNAc, Sialylated LacNAc, LN3, Sialylated LN3, LNnT and LSTc with aModified B. subtilis Strain

In a first step, a B. subtilis strain is modified for sialic acidproduction with genetic knockouts of the nagA, nagB and gamA genes andwith genomic knock-ins of constitutive transcriptional units comprisinggenes encoding the native fructose-6-P-aminotransferase (UniProt IDP0CI73), a glucosamine-6-P-aminotransferase from S. cerevisiae (UniProtID P43577), an N-acetylglucosamine-2-epimerase from B. ovatus (UniProtID A7LVG6) and an N-acetylneuraminate synthase from C. jejuni (UniProtID Q93MP9). In a next step, the mutant strain is further modified withgenomic knock-ins of constitutive transcriptional units comprising genesencoding the N-acylneuraminate cytidylyltransferase NeuA from C. jejuni(UniProt ID Q93MP7), and the alpha-2,6-sialyltransferase PdbST from P.damselae (UniProt ID O66375) to produce 6′-sialyllactose. In a nextstep, the mutant strain is further modified with genomic knock-inscomprising constitutive transcriptional units for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(UniProt ID Q9JXQ6) and an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40 and 41. The novel strain is evaluated for production of amixture of oligosaccharides comprising 6′-SL, LacNAc, sialylated LacNAc,LN3, sialylated LN3, LNnT and LSTc(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experimenton MMsf medium containing lactose as precursor according to the cultureconditions provided in Example 34. After 72 h of incubation, the culturebroth is harvested, and the sugars are analysed on UPLC.

Example 37: Material and Methods Corynebacterium glutamicum

Media

Two different media are used, namely a rich tryptone-yeast extract (TY)medium and a minimal medium for shake flask (MMsf). The minimal mediumuses a 1000x stock trace element mix.

Trace element mix consisted of 10 g/L CaCl₂), 10 g/L FeSO₄·7H₂O, 10 g/LMnSO₄·H₂O, 1 g/L ZnSO₄·7H₂O, 0.2 g/L CuSO₄, 0.02 g/L NiCl₂·6H₂O, 0.2 g/Lbiotin (pH 7.0), and 0.03 g/L protocatechuic acid.

The minimal medium for the shake flasks (MMsf) experiments contained 20g/L (NH₄)₂SO₄, 5 g/L urea, 1 g/L KH₂PO₄, 1 g/L K₂HPO₄, 0.25 g/LMgSO₄·7H₂O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbonsource including but not limited to fructose, maltose, sucrose, glyceroland maltotriose when specified in the examples and 1 ml/L trace elementmix. Depending on the experiment lactose and/or sialic acid could beadded as precursor(s).

The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium),1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven,Belgium). TY agar (TYA) plates consisted of the TY media, with 12 g/Lagar (Difco, Erembodegem, Belgium) added.

Complex medium, e.g., TY, was sterilized by autoclaving (121° C., 21′)and minimal medium by filtration (0.22 μm Sartorius). When necessary,the medium was made selective by adding an antibiotic (e.g., kanamycin,ampicillin).

Strains and Mutations

Corynebacterium glutamicum ATCC 13032, available at the American TypeCulture Collection.

Integrative plasmid vectors based on the Cre/loxP technique as describedby Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 April,67(2):225-33) and temperature-sensitive shuttle vectors as described byOkibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) areconstructed for gene deletions, mutations and insertions. Suitablepromoters for (heterologous) gene expression can be derived from Yim etal. (Biotechnol. Bioeng., 2013 November, 110(11):2959-69). Cloning canbe performed using Gibson Assembly, Golden Gate assembly, Clivaassembly, LCR or restriction ligation.

In an example for the production of LNB, the C. glutamicum strain ismodified with a genomic knock-in of constitutive expression unitscomprising the mutant glmS*54 from E. coli (differing from the wild-typeE. coli glmS, having UniProt ID P17169, by an A39T, an R250C and anG4725 mutation as described by Deng et al. (Biochimie 88, 419-29(2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S.cerevisiae (UniProt ID P43577), one phosphatase chosen from the listcomprising any one or more of the E. coli genes comprising aphA, Cof,HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB,YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG andYbiU or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae orBsAraL from Bacillus subtilis as described in WO 2018122225 and anN-acetylglucosamine beta-1,3-galactosyltransferase chosen from the listcomprising SEQ ID NO: 03, 04, 05, 06, 07, 08, 10, 11, 12 or 13.

In an example for the production of LacNAc, the C. glutamicum strain ismodified with a genomic knock-in of constitutive expression unitscomprising the mutant glmS*54 from E. coli (differing from the wild-typeE. coli glmS, having UniProt ID P17169, by an A39T, an R250C and anG4725 mutation as described by Deng et al. (Biochimie 88, 419-29(2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S.cerevisiae (UniProt ID P43577), one phosphatase chosen from the listcomprising any one or more of the E. coli genes comprising aphA, Cof,HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB,YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG andYbiU or PsMupP from Pseudomonas putida, ScDOG1 from S. cerevisiae orBsAraL from Bacillus subtilis as described in WO 2018122225 and anN-acetylglucosamine beta-1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28,31, 32, 33, 34, 35, 36, 39, 40 and 41. To further fucosylate the LNB orLacNAc, the LNB or LacNAc producing strains are further modified with aconstitutive transcriptional unit for an alpha-1,2-fucosyltransferaselike e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or analpha-1,3-fucosyltransferase like e.g., HpFucT from H. pylori (UniProtID O30511).

In an example for the production of lactose-based oligosaccharides, amutant C. glutamicum strain is created to contain a gene coding for alactose importer (such as e.g., the E. coli lacY with UniProt IDP02920). For 2′FL, 3FL and diFL production, analpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori (GenBankNo. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like e.g., HpFucTfrom H. pylori (UniProt ID O30511) is additionally added to the strain.

In an example for the production of lacto-N-triose (LNT-II, LN3,GlcNAc-b1,3-Gal-b1,4-Glc), the C. glutamicum strain is modified with agenomic knock-in of constitutive transcriptional units comprising alactose importer (such as e.g., the E. coli lacY with UniProt ID P02920)and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g.,LgtA from N. meningitidis (GenBank: AAM33849.1). For LNT production, theLN3 producing strain is further modified with a constitutivetranscriptional unit for an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 03, 04, 05, 06, 07, 08, 10, 11, 12 and 13, that can be delivered tothe strain either via genomic knock-in or from an expression plasmid.For LNnT production, the LN3 producing strain is further modified with aconstitutive transcriptional unit for an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40 and 41. To further fucosylate the LN3, LNT or LNnT, themutant strains are further modified with a constitutive transcriptionalunit for an alpha-1,2-fucosyltransferase like e.g., HpFutC from H.pylori (GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferaselike e.g., HpFucT from H. pylori (UniProt ID O30511).

In an example for sialic acid production, a mutant C. glutamicum strainis created by overexpressing the native fructose-6-P-aminotransferase(UniProt ID Q8NND3) to enhance the intracellular glucosamine-6-phosphatepool. Further on, the enzymatic activities of the C. glutamicum genesldh, cgl2645, nagB, gamA and nagA are disrupted by genetic knockouts anda glucosamine-6-P-aminotransferase from S. cerevisiae (UniProt IDP43577), an N-acetylglucosamine-2-epimerase from B. ovatus (UniProt IDA7LVG6) and an N-acetylneuraminate synthase from C. jejuni (UniProt IDQ931MP9) are overexpressed on the genome. To allow sialylatedoligosaccharide production, the sialic acid producing strain is furthermodified with expression constructs comprising an N-acylneuraminatecytidylyltransferase like e.g., the NeuA enzyme from C. jejuni (UniProtID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV11798.1)or the NeuA enzyme from P. multocida (GenBank No. AMK07891.1), and oneor more copies of a beta-galactoside alpha-2,3-sialyltransferase likee.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-likepolypeptide consisting of amino acid residues 1 to 268 of UniProt IDQ9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity,NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 fromP. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), abeta-galactoside alpha-2,6-sialyltransferase like e.g., PdST6 fromPhotobacterium damselae (UniProt ID O66375) or a PdST6-like polypeptideconsisting of amino acid residues 108 to 497 of UniProt ID O66375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like e.g., from M. musculus (UniProt IDQ64689). In an example for the production of lactose-based sialylatedoligosaccharides, the C. glutamicum mutant strains are further modifiedwith a constitutive transcriptional unit for a lactose importer (such ase.g., the E. coli lacY with UniProt ID P02920).

For growth on sucrose, the mutant strains can additionally be modifiedwith genomic knock-ins of constitutive transcriptional units comprisingthe sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), thefructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and thesucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from thegenome were synthetically synthetized with one of the followingcompanies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivation Conditions

A preculture of 96-well microtiter plate experiments was started from acryovial or a single colony from a TY plate, in 150 μL TY and wasincubated overnight at 37° C. on an orbital shaker at 800 rpm. Thisculture was used as inoculum for a 96-well square microtiter plate, with400 μL MMsf medium by diluting 400x. Each strain was grown in multiplewells of the 96-well plate as biological replicates. These final 96-wellculture plates were then incubated at 37° C. on an orbital shaker at 800rpm for 72 h, or shorter, or longer. At the end of the cultivationexperiment samples were taken from each well to measure the supernatantconcentration (extracellular sugar concentrations, after 5 min. spinningdown the cells), or by boiling the culture broth for 15 min at 60° C.before spinning down the cells (=whole broth concentration, intra- andextracellular sugar concentrations, as defined herein).

Also, a dilution of the cultures was made to measure the optical densityat 600 nm. The cell performance index or CPI was determined by dividingthe oligosaccharide concentrations measured in the whole broth by thebiomass, in relative percentages compared to the reference strain. Thebiomass is empirically determined to be approximately ⅓rd of the opticaldensity measured at 600 nm.

Example 38: Production of 2′FLNB with a Modified C. glutamicum Strain

A C. glutamicum strain is first modified for LNB production and growthon sucrose by genomic knock-out of the ldh, cgl2645 and nagB genes andgenomic knock-ins of constitutive transcriptional units comprising genesencoding the mutant glmS*54 from E. coli (differing from the wild-typeE. coli glmS, having UniProt ID P17169, by an A39T, an R250C and anG472S mutation as described by Deng et al. (Biochimie 88, 419-29(2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S.cerevisiae (UniProt ID P43577), the phosphatase AraL from B. subtilis(UniProt ID P94526), the N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 03, 04, 05, 06, 07, 08, 10, 11, 12 and 13, the sucrose transporter(CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk)from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP)from B. adolescentis (UniProt ID A0ZZH6). In a next step, the LNBproducing strain is transformed with an expression plasmid comprising aconstitutive transcriptional unit for the alpha-1,2-fucosyltransferaseHpFutC from H. pylori (GenBank No. AAD29863.1). The novel strain isevaluated for the production 2′FLNB in a growth experiment on MMsfmedium lacking a precursor according to the culture conditions providedin Example 37. After 72 h of incubation, the culture broth is harvested,and the sugars are analysed on UPLC.

Example 39: Production of a Mixture Comprising Sialylated LacNAc with aModified C. glutamicum Strain

A C. glutamicum strain is first modified for LacNAc production andgrowth on sucrose by genomic knock-out of the ldh, cgl2645, nagB, nagAand gamA genes and genomic knock-ins of constitutive transcriptionalunits comprising genes encoding the native fructose-6-P-aminotransferase(UniProt ID Q8NND3), the mutant glmS*54 from E. coli (differing from thewild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250Cand an G4725 mutation as described by Deng et al. (Biochimie 88, 419-29(2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S.cerevisiae (UniProt ID P43577), the phosphatase AraL from B. subtilis(UniProt ID P94526), the N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40 and 41, the sucrose transporter (CscB) from E. coli W(UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProtID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis(UniProt ID A0ZZH6). In a next step for sialic acid synthesis, themutant strain was further modified with genomic knock-ins ofconstitutive transcriptional units comprising genes encoding theN-acetylglucosamine-2-epimerase from B. ovatus (UniProt ID A7LVG6), andthe N-acetylneuraminate synthase from C. jejuni (UniProt ID Q93MP9). Ina next step, the novel strain is transformed with an expression plasmidcomprising constitutive transcriptional units comprising the geneencoding the NeuA enzyme from C. jejuni (UniProt ID Q93MP7) combinedwith the gene encoding either the beta-galactosidealpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt IDQ9CLP3) or the beta-galactoside alpha-2,6-sialyltransferase PdST6 fromP. damselae (UniProt ID O66375). The novel strains are evaluated forproduction of LacNAc, sialic acid and sialylated LacNAc in a growthexperiment on MMsf medium lacking a precursor according to the cultureconditions provided in Example 37. When adding lactose as precursor tothe MMsf medium, the mutant strains are also evaluated for additionalproduction of 3′-SL or 6′-SL, depending on the alpha-sialyltransferaseexpressed. After 72 h of incubation, the culture broth is harvested, andthe sugars are analysed on UPLC.

Example 40: Materials and Methods Chlamydomonas reinhardtii

Media

C. reinhardtii cells were cultured in Tris-acetate-phosphate (TAP)medium (pH 7.0). The TAP medium uses a 1000x stock Hutner's traceelement mix. Hutner's trace element mix consisted of 50 g/L Na₂EDTA·H₂O(Titriplex III), 22 g/L ZnSO₄·7H₂O, 11.4 g/L H₃BO₃, 5 g/L MnCl₂·4H₂O, 5g/L FeSO₄·7H₂O, 1.6 g/L CoCl₂·6H₂O, 1.6 g/L CuSO₄·5H₂O and 1.1 g/L(NH₄)₆MoO₃.

The TAP medium contained 2.42 g/L Tris(tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108g/L K₂HPO₄, 0.054 g/L KH₂PO₄ and 1.0 mL/L glacial acetic acid. The saltstock solution consisted of 15 g/L NH₄Cl, 4 g/L MgSO₄·7H₂O and 2 g/LCaCl₂·2H₂O. As precursor for saccharide synthesis, precursors like e.g.,galactose, glucose, fructose and/or fucose could be added. Medium wassterilized by autoclaving (121° C., 21′). For stock cultures on agarslants TAP medium was used containing 1% agar (of purified highstrength, 1000 g/cm²).

Strains, Plasmids and Mutations

C. reinhardtii wild-type strains 21 gr (CC-1690, wild-type, mt+), 6145C(CC-1691, wild-type, mt−), CC-125 (137c, wild-type, mt+), CC-124 (137c,wild-type, mt−) as available from Chlamydomonas Resource Center(www.chlamycollection.org), University of Minnesota, U.S.A.

Expression plasmids originated from pSI103, as available fromChlamydomonas Resource Center. Cloning can be performed using GibsonAssembly, Golden Gate assembly, Cliva assembly, LCR or restrictionligation. Suitable promoters for (heterologous) gene expression can bederived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142).Targeted gene modification (like gene knock-out or gene replacement) canbe carried using the Crispr-Cas technology as described e.g., by Jianget al. (Eukaryotic Cell 2014, 13(11): 1465-1469).

Transformation via electroporation was performed as described by Wang etal. (Biosci. Rep. 2019, 39: BSR2018210). Cells were grown in liquid TAPmedium under constant aeration and continuous light with a lightintensity of 8000 Lx until the cell density reached 1.0-2.0×10⁷cells/mL. Then, the cells were inoculated into fresh liquid TAP mediumin a concentration of 1.0×10⁶ cells/mL and grown under continuous lightfor 18-20 h until the cell density reached 4.0×10⁶ cells/mL. Next, cellswere collected by centrifugation at 1250 g for 5 min at roomtemperature, washed and resuspended with pre-chilled liquid TAP mediumcontaining 60 mM sorbitol (Sigma, U.S.A.), and iced for 10 min. Then,250 μL of cell suspension (corresponding to 5.0×10⁷ cells) were placedinto a pre-chilled 0.4 cm electroporation cuvette with 100 ng plasmidDNA (400 ng/mL). Electroporation was performed with 6 pulses of 500 Veach having a pulse length of 4 ms and pulse interval time of 100 msusing a BTX ECM830 electroporation apparatus (1575 Ω, 50 gD). Afterelectroporation, the cuvette was immediately placed on ice for 10 min.Finally, the cell suspension was transferred into a 50 ml conicalcentrifuge tube containing 10 mL of fresh liquid TAP medium with 60 mMsorbitol for overnight recovery at dim light by slowly shaking. Afterovernight recovery, cells were recollected and plated with starchembedding method onto selective 1.5% (w/v) agar-TAP plates containingampicillin (100 mg/L) or chloramphenicol (100 mg/L). Plates were thenincubated at 23+/−0.5° C. under continuous illumination with a lightintensity of 8000 Lx. Cells were analysed 5-7 days later.

In an example for production of UDP-galactose, C. reinhardtii cells aremodified with transcriptional units comprising the gene encoding thegalactokinase from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and thegene encoding the UDP-sugar pyrophosphorylase (USP) from A. thaliana(UniProt ID Q9C511).

In an example for production of LNB, C. reinhardtii cells modified forUDP-galactose production are further modified with an expression plasmidcomprising a transcriptional unit for the N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 03, 04, 05, 06, 07, 08, 10, 11, 12, and 13. Additionally, themutant C. reinhardtii cells can be modified with an expression plasmidcomprising a transcriptional unit for an alpha-1,2-fucosyltransferaselike e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or analpha-1,3-fucosyltransferase like e.g., HpFucT from H. pylori (UniProtID O30511).

In an example for production of LacNAc, C. reinhardtii cells modifiedfor UDP-galactose production are further modified with an expressionplasmid comprising a transcriptional unit for the N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 31, 32, 33, 34, 35,36, 39, 40, and 41. Additionally, the mutant C. reinhardtii cells can bemodified with an expression plasmid comprising a transcriptional unitfor an alpha-1,2-fucosyltransferase like e.g., HpFutC from H. pylori(GenBank No. AAD29863.1) and/or an alpha-1,3-fucosyltransferase likee.g., HpFucT from H. pylori (UniProt ID O30511).

In an example for CMP-sialic acid synthesis, C. reinhardtii cells aremodified with constitutive transcriptional units for anUDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase likee.g., GNE from Homo sapiens (UniProt ID Q9Y223) or a mutant form of thehuman GNE polypeptide comprising the R263L mutation, anN-acylneuraminate-9-phosphate synthetase like e.g., NANS from Homosapiens (UniProt ID Q9NR45) and an N-acylneuraminatecytidylyltransferase like e.g., CMAS from Homo sapiens (UniProt IDQ8NFW8). In an example for production of sialylated oligosaccharides, C.reinhardtii cells are modified with a CMP-sialic acid transporter likee.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localizedsialyltransferase chosen from species like e.g., Homo sapiens, Musmusculus, Rattus norvegicus.

Heterologous and Homologous Expression

Genes that needed to be expressed, be it from a plasmid or from thegenome were synthetically synthetized with one of the followingcompanies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivation Conditions

Cells of C. reinhardtii were cultured in selective TAP-agar plates at23+/−0.5° C. under 14/10 h light/dark cycles with a light intensity of8000 Lx. Cells were analysed after 5 to 7 days of cultivation.

For high-density cultures, cells could be cultivated in closed systemslike e.g., vertical or horizontal tube photobioreactors, stirred tankphotobioreactors or flat panel photobioreactors as described by Chen etal. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al.(Biotechnol. Prog. 2018, 34: 811-827).

Example 41: Production of LNB and 2′FLNB in Modified C. reinhardtiiCells

C. reinhardtii cells are engineered as described in Example 40 forproduction of UDP-Gal with genomic knock-ins of constitutivetranscriptional units comprising the galactokinase from A. thaliana(KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) fromA. thaliana (UniProt ID Q9C5I1). In a next step, the mutant cells aretransformed with an expression plasmid comprising transcriptional unitscomprising an N-acetylglucosamine beta-1,3-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 03, 04, 05, 06, 07, 08, 10, 11, 12and 13 and the alpha-1,2-fucosyltransferase HpFutC from H. pylori(GenBank No. AAD29863.1). The novel strain is evaluated in a cultivationexperiment on TAP-agar plates comprising galactose as precursoraccording to the culture conditions provided in Example 40. After 5 daysof incubation, the cells are harvested, and the production of LNB and2′FLNB is analyzed on UPLC.

Example 42: Production of LacNAc and 3′-Fucosylated LacNAc in ModifiedC. reinhardtii Cells

C. reinhardtii cells are engineered as described in Example 40 forproduction of UDP-Gal with genomic knock-ins of constitutivetranscriptional units comprising the galactokinase from A. thaliana(KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) fromA. thaliana (UniProt ID Q9C5I1). In a next step, the mutant cells aretransformed with an expression plasmid comprising transcriptional unitscomprising an N-acetylglucosamine beta-1,4-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23,26, 27, 28, 31, 32, 33, 34, 35, 36, 39, 40 and 41 and thealpha-1,3-fucosyltransferase like e.g., HpFucT from H. pylori (UniProtID O30511). The novel strain is evaluated in a cultivation experiment onTAP-agar plates comprising galactose as precursor according to theculture conditions provided in Example 40. After 5 days of incubation,the cells are harvested, and the production of LacNAc and 3′-fucosylatedLacNAc is analyzed on UPLC.

Example 43: Materials and Methods Animal Cells

Isolation of Mesenchymal Stem Cells from Adipose Tissue of DifferentMammals

Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle,pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction(e.g., in case of humans, after informed consent) and kept in phosphatebuffer saline supplemented with antibiotics. Enzymatic digestion of theadipose tissue is performed followed by centrifugation to isolatemesenchymal stem cells. The isolated mesenchymal stem cells aretransferred to cell culture flasks and grown under standard growthconditions, e.g., 37° C., 5% CO₂. The initial culture medium includesDMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% foetalbovine serum), and 1% antibiotics. The culture medium is subsequentlyreplaced with 10% FBS (foetal bovine serum)-supplemented media after thefirst passage. For example, Ahmad and Shakoori (2013, Stem Cell Regen.Med. 9(2): 29-36), which is incorporated herein by reference in itsentirety for all purposes, describes certain variation(s) of themethod(s) described herein in this example.

Isolation of Mesenchymal Stem Cells from Milk

This example illustrates isolation of mesenchymal stem cells from milkcollected under aseptic conditions from human or any other mammal(s)such as described herein. An equal volume of phosphate buffer saline isadded to diluted milk, followed by centrifugation for 20 min. The cellpellet is washed thrice with phosphate buffer saline and cells areseeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM mediumsupplemented with 10% fetal bovine serum and 1% antibiotics understandard culture conditions. For example, Hassiotou et al. (2012, StemCells. 30(10): 2164-2174), which is incorporated herein by reference inits entirety for all purposes, describes certain variation(s) of themethod(s) described herein in this example.

Differentiation of Stem Cells Using 2D and 3D Culture Systems

The isolated mesenchymal cells can be differentiated into mammary-likeepithelial and luminal cells in 2D and 3D culture systems. See, forexample, Huynh et al. 1991. Exp Cell Res. 197(2): 191-199; Gibson et al.1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al.1999; Animal Cell Technology: Basic & Applied Aspects, Springer,Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3):26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5):C348-C356.

For 2D culture, the isolated cells were initially seeded in cultureplates in growth media supplemented with 10 ng/ml epithelial growthfactor and 5 pg/ml insulin. At confluence, cells were fed with growthmedium supplemented with 2% fetal bovine serum, 1%penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin),and 5 pg/ml insulin for 48h. To induce differentiation, the cells werefed with complete growth medium containing 5 pg/ml insulin, 1 pg/mlhydrocortisone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone, and 1pg/ml prolactin. After 24 h, serum is removed from the completeinduction medium.

For 3D culture, the isolated cells were trypsinized and cultured inMatrigel, hyaluronic acid, or ultra-low attachment surface cultureplates for six days and induced to differentiate and lactate by addinggrowth media supplemented with 10 ng/ml epithelial growth factor and 5pg/ml insulin. At confluence, cells were fed with growth mediumsupplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U/ml penicillin, 100 ug/ml streptomycin), and 5 pg/ml insulin for 48 h.To induce differentiation, the cells were fed with complete growthmedium containing 5 pg/ml insulin, 1 pg/ml hydrocortisone, 0.65 ng/mltriiodothyronine, 100 nM dexamethasone, and 1 pg/ml prolactin. After24h, serum is removed from the complete induction medium.

Method of Making Mammary-Like Cells

Mammalian cells are brought to induced pluripotency by reprogrammingwith viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc. Theresultant reprogrammed cells are then cultured in Mammocult media(available from Stem Cell Technologies), or mammary cell enrichmentmedia (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone,insulin, EGF) to make them mammary-like, from which expression of selectmilk components can be induced. Alternatively, epigenetic remodellingare performed using remodelling systems such as CRISPR/Cas9, to activateselect genes of interest, such as casein, a-lactalbumin to beconstitutively on, to allow for the expression of their respectiveproteins, and/or to down-regulate and/or knock-out select endogenousgenes as described e.g., in WO 2021067641, which is incorporated hereinby reference in its entirety for all purposes.

Cultivation

Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA,1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, and 5 pg/mlhydrocortisone. Completed lactation media includes high glucoseDMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, 5pg/ml hydrocortisone, and 1 pg/ml prolactin (5 ug/ml in Hyunh 1991).Cells are seeded at a density of 20,000 cells/cm′ onto collagen coatedflasks in completed growth media and left to adhere and expand for 48hours in completed growth media, after which the media is switched outfor completed lactation media. Upon exposure to the lactation media, thecells start to differentiate and stop growing. Within about a week, thecells start secreting lactation product(s) such as milk lipids, lactose,casein and whey into the media. A desired concentration of the lactationmedia can be achieved by concentration or dilution by ultrafiltration. Adesired salt balance of the lactation media can be achieved by dialysis,for example, to remove unwanted metabolic products from the media.Hormones and other growth factors used can be selectively extracted byresin purification, for example, the use of nickel resins to removeHis-tagged growth factors, to further reduce the levels of contaminantsin the lactated product.

Example 44: Evaluation of 2′FL, LNFP-I and 2′FLNB Production in aNon-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells asdescribed in Example 43 are modified via CRISPR-CAS to over-express acodon-optimized N-acetylglucosamine beta-1,3-galactosyltransferasechosen from the list comprising SEQ ID NOs:03, 04, 05, 06, 07, 08, 10,11, 12 and 13, the GDP-fucose synthase GFUS from Homo sapiens (UniProtID Q13630), and a codon-optimized alpha-1,2-fucosyltransferase from H.pylori (GenBank No. AAD29863.1). Cells are seeded at a density of 20,000cells/cm′ onto collagen coated flasks in completed growth media and leftto adhere and expand for 48 hours in completed growth media, after whichthe media is switched out for completed lactation media for about 7days. After cultivation as described in Example 43, cells are subjectedto UPLC to analyse for production of 2′FL, LNFP-I (lacto-N-fucopentaoseI, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and 2′FLNB.

Example 45: Evaluation of LacNAc, Sialylated LacNAc and Sialyl-Lewis xProduction in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells asdescribed in Example 43 are modified via CRISPR-CAS to over-express anN-acetylglucosamine beta-1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28,31, 32, 33, 34, 35, 36, 39, 40 and 41, the GDP-fucose synthase GFUS fromHomo sapiens (UniProt ID Q13630), the galactosidealpha-1,3-fucosyltransferase FUT3 from Homo sapiens (UniProt ID P21217),the N-acylneuraminate cytidylyltransferase from Mus musculus (UniProt IDQ99KK2) and the CMP-N-acetylneuraminate-beta-1,4-galactosidealpha-2,3-sialyltransferase ST3GAL3 from Homo sapiens (UniProt IDQ11203). All genes introduced in the cells are codon-optimized to thehost. Cells are seeded at a density of 20,000 cells/cm² onto collagencoated flasks in completed growth media and left to adhere and expandfor 48 hours in completed growth media, after which the media isswitched out for completed lactation media for about 7 days. Aftercultivation as described in Example 43, cells are subjected to UPLC toanalyse for production of LacNAc, sialylated LacNAc and sialyl-Lewis x.

1.-108. (canceled)
 109. A method of producing an oligosaccharide ordisaccharide having an N-acetylglucosamine unit at the reducing end, themethod comprising: a. providing a cell which is capable of: (i)synthesizing a nucleotide-sugar and the monosaccharideN-acetylglucosamine (GlcNAc), and (ii) expressing a glycosyltransferaseto glycosylate the GlcNAc monosaccharide to produce the disaccharide oroligosaccharide, b. cultivating the cell under conditions permissive forproducing the disaccharide or oligosaccharide, and c. optionally,separating the disaccharide or oligosaccharide from the cultivation.110. The method according to claim 109, wherein the cell furtherproduces one or more lactose-based mammalian milk oligosaccharides(MMOs), thereby producing a mixture comprising a disaccharide and/oroligosaccharide having an N-acetylglucosamine unit at the reducing end.111. The method according to claim 109, wherein the cell expresses atleast one glucosamine 6-phosphate N-acetyltransferase and a phosphataseto synthesize the monosaccharide N-acetylglucosamine.
 112. The methodaccording to claim 109, wherein the cell expresses at least oneglycosyltransferase to glycosylate N-acetylglucosamine.
 113. The methodaccording to claim 109, wherein the cell is genetically modified toproduce the disaccharide or oligosaccharide.
 114. The method accordingto claim 113, wherein the cell is modified with one or more geneexpression modules, wherein the expression from any of the geneexpression modules is either constitutive or is created by a naturalinducer.
 115. The method according to claim 113, wherein the cellcomprises multiple copies of the same coding DNA sequence encoding oneprotein.
 116. The method according to claim 109, wherein the cell ismodified in the expression or activity of an enzyme selected from thegroup consisting of glucosamine 6-phosphate N-acetyltransferase,phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.
 117. The method accordingto claim 109, wherein the nucleotide-sugar is selected from the groupconsisting of UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine(UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc),UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose(GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, and UDP-xylose.
 118. The method according to claim 109,wherein the nucleotide-sugar is UDP-galactose and theglycosyltransferase is an N-acetylglucosamineb-1,3-galactosyltransferase or an N-acetylglucosamineb-1,4-galactosyltransferase.
 119. The method according to claim 109,wherein the oligosaccharide has a lacto-N-biose (Gal-b1,3-GlcNAc) or anN-acetyllactosamine (Gal-b1,4-GlcNAc) at the reducing end.
 120. Themethod according to claim 118, wherein the N-acetylglucosamineb-1,3-galactosyltransferase is a glycosyltransferase having a. PFAMdomain PF00535, and i. comprises the peptide of SEQ ID NO:1, ii.comprises the peptide of SEQ ID NO:2, iii. comprises a polypeptidesequence of any one of SEQ ID NO:3, 4, 5, 6, 7 or 8, iv. is a functionalhomologue, variant, or derivative of any one of SEQ ID NO:3, 4, 5, 6, 7or 8, having at least 80% overall sequence identity to the full-lengthof any one of the N-acetylglucosamine b-1,3-galactosyltransferasepolypeptide of SEQ ID NO:3, 4, 5, 6, 7 or 8, and havingN-acetylglucosamine b-1,3-galactosyltransferase activity, v. comprisesan oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 consecutive amino acid residues from any one of SEQ IDNO:3, 4, 5, 6, 7 or 8, and having N-acetylglucosamineb-1,3-galactosyltransferase activity, vi. is a functional fragment ofany one of SEQ ID NO:3, 4, 5, 6, 7 or 8, and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or vii. comprises a polypeptidecomprising an peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NO:3, 4, 5, 6, 7 or 8, andhaving N-acetylglucosamine b-1,3-galactosyltransferase activity, or b.PFAM domain IPR002659, and i. comprises the peptide of SEQ ID NO:9, orii. comprises a polypeptide sequence of any one of SEQ ID NO:10, 11, 12or 13, or iii. is a functional homologue, variant or derivative of anyone of SEQ ID NO:10, 11, 12 or 13 having at least 80% overall sequenceidentity to the full-length of any one of the N-acetylglucosamineb-1,3-galactosyltransferase polypeptide of SEQ ID NO:10, 11, 12 or 13and having N-acetylglucosamine b-1,3-galactosyltransferase activity, oriv. comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive amino acid residues from anyone of SEQ ID NO:10, 11, 12 or 13 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or v. is a functional fragment ofany one of SEQ ID NO:10, 11, 12 or 13 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or vi. comprises a polypeptidecomprising an peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NO:10, 11, 12 or 13 and havingN-acetylglucosamine b-1,3-galactosyltransferase activity.
 121. Themethod according to claim 118, wherein the N-acetylglucosamineb-1,4-galactosyltransferase is a glycosyltransferase comprising a. PFAMdomain PF01755, and i. comprises the peptide of SEQ ID NO:14, ii.comprises a polypeptide sequence of any one of SEQ ID NO:15, 16, 17, 18,19, 20, 21, 22 or 23, iii. is a functional homologue, variant orderivative of any one of SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22 or 23,having at least 80% overall sequence identity to the full-length of anyone of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptideof SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22 or 23, and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, iv. comprisesan oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 consecutive amino acid residues from any one of SEQ IDNO:15, 16, 17, 18, 19, 20, 21, 22 or 23, and having N-acetylglucosamineb-1,4-galactosyltransferase activity, v. is a functional fragment of anyone of SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22 or 23, and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or vi.comprises a polypeptide comprising an peptide having at least 80%sequence identity to the full-length peptide of any one of SEQ ID NO:15,16, 17, 18, 19, 20, 21, 22 or 23, and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or b. PFAM domain PF00535, and i.comprises the peptide of SEQ ID NO:24, ii. comprises the peptide of SEQID NO:25, iii. comprises a polypeptide sequence of any one of SEQ IDNO:26, 27 or 28, or iv. is a functional homologue, variant or derivativeof any one of SEQ ID NO:26, 27 or 28 having at least 80% overallsequence identity to the full-length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide of SEQ IDNO:26, 27 or 28 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, v. comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20consecutive amino acid residues from any one of SEQ ID NO:26, 27 or 28and having N-acetylglucosamine b-1,4-galactosyltransferase activity, vi.is a functional fragment of any one of SEQ ID NO:26, 27 or 28 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or vii.comprises a polypeptide comprising an peptide having at least 80%sequence identity to the full-length peptide of any one of SEQ ID NO:26,27 or 28 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, or c. PFAM domain PF02709 and not having PFAM domain PF00535,and i. comprises the peptide of SEQ ID NO:29, ii. comprises the peptideof SEQ ID NO:30, iii. comprises a polypeptide sequence of any one of SEQID NO:31, 32, 33, 34, 35 or 36, iv. is a functional homologue, variantor derivative of any one of SEQ ID NO:31, 32, 33, 34, 35 or 36 having atleast 80% overall sequence identity to the full-length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNO:31, 32, 33, 34, 35 or 36 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, v. comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20consecutive amino acid residues from any one of SEQ ID NO:31, 32, 33,34, 35 or 36 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, or vi. is a functional fragment of any one of SEQ ID NO:31,32, 33, 34, 35 or 36 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or vii. comprises a polypeptidecomprising an peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NO:31, 32, 33, 34, 35 or 36 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or d.PFAM domain PF03808, and i. comprises the peptide of SEQ ID NO:37, ii.comprises the peptide of SEQ ID NO:38, iii. comprises a polypeptidesequence of any one of SEQ ID NO:39, 40 or 41, or iv. is a functionalhomologue, variant or derivative of any one of SEQ ID NO:39, 40 or 41having at least 80% overall sequence identity to the full-length of anyone of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptidewith SEQ ID NO:39, 40 or 41 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, v. comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20consecutive amino acid residues from any one of SEQ ID NO:39, 40 or 41and having N-acetylglucosamine b-1,4-galactosyltransferase activity, vi.is a functional fragment of any one of SEQ ID NO:39, 40 or 41 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or vii.comprises a polypeptide comprising an peptide having at least 80%sequence identity to the full-length peptide of any one of SEQ ID NO:39,40 or 41 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity.
 122. The method according to claim 116, wherein a. theglucosamine 6-phosphate N-acetyltransferase is a polypeptide sequencecomprising the polypeptide of UniProt ID P43577 or is a functionalhomologue, variant or derivative of the polypeptide of UniProt ID P43577having at least 80% overall sequence identity to the full-length of thepolypeptide of UniProt ID P43577 and having glucosamine 6-phosphateN-acetyltransferase activity, and b. theL-glutamine-D-fructose-6-phosphate aminotransferase is a polypeptidesequence comprising the polypeptide of UniProt ID P17169 or is afunctional homologue, variant or derivative of the polypeptide ofUniProt ID P17169 having at least 80% overall sequence identity to thefull-length of the polypeptide of UniProt ID P17169 and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity; or is amodified version that differs from the polypeptide of UniProt ID P17169by an A39T, an R250C and an G472S mutation.
 123. The method according toclaim 109, wherein the cell is capable of catabolizing a carbon sourceselected from the group consisting of glucose, fructose, galactose,lactose, sucrose, maltose, malto-oligosaccharides, maltotriose,sorbitol, xylose, rhamnose, mannose, methanol, ethanol, arabinose,trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor,high-fructose syrup, molasses, glycerol, acetate, citrate, lactate, andpyruvate.
 124. The method according to claim 109, wherein the cell isunable to convert N-acetylglucosamine-6-phosphate toglucosamine-6-phosphate, and/or unable to convertglucosamine-6-phosphate to fructose-6-phoshate.
 125. The methodaccording to claim 109, wherein the cell is modified to produceGDP-fucose, wherein the modification comprises: knock-out of anUDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferaseencoding gene, over-expression of a GDP-L-fucose synthase encoding gene,over-expression of a GDP-mannose 4,6-dehydratase encoding gene,over-expression of a mannose-1-phosphate guanylyltransferase encodinggene, over-expression of a phosphomannomutase encoding gene, orover-expression of a mannose-6-phosphate isomerase encoding gene. 126.The method according to claim 109, wherein the cell is modified toproduce UDP-galactose, wherein the modification comprises: knock-out ofa 5′-nucleotidase/UDP-sugar hydrolase encoding gene, or knock-out of agalactose-1-phosphate uridylyltransferase encoding gene.
 127. The methodaccording to claim 109, wherein the cell is modified to produceCMP-N-acetylneuraminic acid, wherein the modification comprises:over-expression of an CMP-sialic acid synthetase encoding gene,over-expression of a sialate synthase encoding gene, or over-expressionof an N-acetyl-D-glucosamine 2-epimerase encoding gene.
 128. The methodaccording to claim 109, wherein the cell is capable of expressing atleast one further glycosyltransferase, wherein the furtherglycosyltransferase is selected from the group consisting of afucosyltransferase, sialyltransferase, galactosyltransferase,glucosyltransferase, mannosyltransferase,N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase,N-acetylmannosaminyltransferase, xylosyltransferase,glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase,N-glycolylneuraminyltransferase, rhamnosyltransferase,N-acetylrhamnosyltransferase,UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminase,UDP-N-acetylglucosamine enolpyruvyl transferase andfucosaminyltransferase. and optionally, the cell is modified in theexpression or activity of the further glycosyltransferase.
 129. Themethod according to claim 109, wherein the cell uses one or moreprecursor(s) to produce the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end, the precursor(s) being fed to the cellfrom the cultivation medium, or the cell produces one or moreprecursor(s) to produce the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end.
 130. The method according to claim 129,wherein the precursor to produce the disaccharide or oligosaccharide iscompletely converted into the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end.
 131. The method according to claim 109,wherein the cell produces the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end intracellularly and wherein a fractionor substantially all of the produced disaccharide or oligosaccharidehaving a GlcNAc unit at the reducing end remains intracellularly and/oris excreted outside the cell via passive or active transport.
 132. Themethod according to claim 109, wherein the cell expresses a membranetransporter protein having transport activity and transports compoundsacross an outer membrane of the cell.
 133. The method according to claim132, wherein the membrane transporter having transport activity:controls the flow over the outer membrane of the disaccharide oroligosaccharide having a GlcNAc unit at the reducing end and/or of oneor more precursor(s) and/or acceptor(s) to be used in the production ofthe disaccharide or oligosaccharide having a GlcNAc unit at the reducingend, and/or provides improved production and/or enabled and/or enhancedefflux of the disaccharide or oligosaccharide having a GlcNAc unit atthe reducing end.
 134. The method according to claim 109, wherein thecell comprises a modification for reduced production of acetate comparedto a non-modified progenitor, optionally the cell comprises a lower orreduced expression, and/or abolished, impaired, reduced, or delayedactivity of at least one of a beta-galactosidase, galactosideO-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase,glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor,ribonucleotide monophosphatase, EIICBA-Nag,UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase,L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase,N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase,EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphateuridylyltransferase, glucose-1-phosphate adenylyltransferase,glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1,ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphateisomerase, aerobic respiration control protein, transcriptionalrepressor IclR, lon protease, glucose-specific translocatingphosphotransferase enzyme IIBC component ptsG, glucose-specifictranslocating phosphotransferase (PTS) enzyme IIBC component malX,enzyme IIA^(Glc), beta-glucoside specific PTS enzyme II,fructose-specific PTS multiphosphoryl transfer protein FruA and FruB,ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase,acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, orpyruvate decarboxylase compared to a non-modified progenitor.
 135. Themethod according to claim 109, wherein the cell is capable of producingphosphoenolpyruvate (PEP) and/or the cell is modified for enhancedproduction and/or supply of PEP compared to a non-modified progenitor.136. The method according to claim 109, wherein the cell comprises acatabolic pathway for a selected monosaccharide, disaccharide oroligosaccharide which is at least partially inactivated, themonosaccharide, disaccharide, or oligosaccharide being involved inand/or required to produce the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end.
 137. The method according to claim 109,wherein the cell resists lactose killing when grown in an environment inwhich lactose is combined with one or more other carbon source(s). 138.The method according to claim 109, wherein the cell produces 90 g/L ormore of the disaccharide or oligosaccharide having a GlcNAc at thereducing end in the whole broth and/or supernatant, and/or wherein thedisaccharide or oligosaccharide having a GlcNAc at the reducing end inthe whole broth and/or supernatant has a purity of at least 80% measuredon the total amount of the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end and its precursor(s) in the whole brothand/or supernatant, respectively.
 139. The method according to claim109, wherein the conditions comprise: use of a culture medium comprisingat least one precursor and/or acceptor to produce the disaccharide oroligosaccharide having a GlcNAc unit at the reducing end, and/or addingto the culture medium at least one precursor and/or acceptor feed toproduce the disaccharide or oligosaccharide having a GlcNAc unit at thereducing end.
 140. The method according to claim 109, wherein theculture medium contains at least one precursor selected from the groupconsisting of lactose, galactose, fucose, and sialic acid.
 141. Themethod according to claim 109, wherein a first phase of exponential cellgrowth is provided by adding a carbon-based substrate to the culturemedium comprising a precursor, followed by a second phase wherein: onlya carbon-based substrate is added to the culture medium, or acarbon-based substrate and a precursor are added to the culture medium.142. The method according to claim 109, wherein the cell produces amixture of charged and/or neutral disaccharides and oligosaccharidescomprising at least one disaccharide or oligosaccharide having a GlcNAcunit at the reducing end.
 143. The method according to claim 109,wherein the cell produces a mixture of charged and/or neutraloligosaccharides comprising at least oligosaccharide having a GlcNAcunit at the reducing end.
 144. The method according to claim 109,wherein the oligosaccharide is selected from the group consisting of2-fucosyl lacto-N-biose, 4-fucosyl lacto-N-biose, 2-4-difucosyllacto-N-biose, 3′-sialyl lacto-N-biose, 6′-sialyl lacto-N-biose,3′,6′-disialyl lacto-N-biose, 6,6′-disialyl lacto-N-biose,2′-fucosyl-3′-sialyl lacto-N-biose, 2′-fucosyl-6′-sialyl lacto-N-biose,4-fucosyl-3′-sialyl lacto-N-biose, 4-fucosyl-6′-sialyl lacto-N-biose,2-fucosyl N-acetyllactosamine, 3′-fucosyl N-acetyllactosamine,2,3′-difucosyl N-acetyllactosamine, 3′-sialyl N-acetyllactosamine,6′-sialyl N-acetyllactosamine, 3′,6′-disialyl N-acetyllactosamine,6,6′-disialyl N-acetyllactosamine, 2′-fucosyl-3′-sialylN-acetyllactosamine, 2′-fucosyl-6′-sialyl N-acetyllactosamine,3-fucosyl-3 ‘-sialyl N-acetyllactosamine, 3’-fucosyl-6′-sialylN-acetyllactosamine, P1 trisaccharide (Gal-a1,4-Gal-b1,4-GlcNAc), thexenotransplantation epitope (Gal-a1,3-Gal-b1,4-GlcNAc),Gal-b14-(Galb13)-GlcNAc, poly-N-acetyllactosamine, andGalNAc-b1,3-Gal-b1,4-GlcNAc.
 145. The method according to claim 109,wherein the disaccharide having a GlcNAc unit at the reducing end doesnot comprise chitobiose (GlcNAc-GlcNAc).
 146. The method according toclaim 109, wherein the oligosaccharide having a GlcNAc unit at thereducing end does not comprise a chitobiose at the reducing end.
 147. Ametabolically engineered cell for producing an oligosaccharide ordisaccharide having an N-acetylglucosamine unit at the reducing end,wherein the cell is capable of: (i) synthesizing a nucleotide-sugar andthe monosaccharide N-acetylglucosamine (GlcNAc) and (ii) expressing aglycosyltransferase to glycosylate the GlcNAc monosaccharide to producethe disaccharide or oligosaccharide.
 148. The metabolically engineeredcell of claim 147, wherein the cell further produces one or morelactose-based mammalian milk oligosaccharides (MMOs), thereby producinga mixture comprising a disaccharide and/or oligosaccharide having anN-acetylglucosamine unit at the reducing end.
 149. The metabolicallyengineered cell of claim 147, wherein the cell is metabolicallyengineered to produce the disaccharide or oligosaccharide having anN-acetylglucosamine at the reducing end.
 150. The metabolicallyengineered cell of claim 147, wherein the cell is metabolicallyengineered to produce the oligosaccharide having an N-acetylglucosamineat the reducing end.
 151. The metabolically engineered cell of claim147, wherein the cell is modified with one or more gene expressionmodules, wherein the expression from any of the gene expression modulesis either constitutive or is created by a natural inducer.
 152. Themetabolically engineered cell of claim 147, wherein the cell comprisesmultiple copies of the same coding DNA sequence encoding one protein.153. The metabolically engineered cell of claim 147, wherein the cellexpresses at least one glucosamine 6-phosphate N-acetyltransferase and aphosphatase to synthesize N-acetylglucosamine.
 154. The metabolicallyengineered cell of claim 147, wherein the cell expresses at least oneglycosyltransferase to glycosylate N-acetylglucosamine.
 155. Themetabolically engineered cell of claim 147, wherein the cell is modifiedin the expression or activity of an enzyme selected from the groupconsisting of glucosamine 6-phosphate N-acetyltransferase, phosphatase,glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.
 156. The metabolicallyengineered cell of claim 147, wherein the nucleotide-sugar is selectedfrom the group consisting of UDP-galactose (UDP-Gal),UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine(UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose(GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, and UDP-xylose.
 157. The metabolically engineered cell ofclaim 147, wherein the nucleotide-sugar is UDP-galactose and theglycosyltransferase is an N-acetylglucosamineb-1,3-galactosyltransferase or an N-acetylglucosamineb-1,4-galactosyltransferase.
 158. The metabolically engineered cell ofclaim 147, wherein the oligosaccharide has a lacto-N-biose(Gal-b1,3-GlcNAc) or an N-acetyllactosamine (Gal-b1,4-GlcNAc) at thereducing end.
 159. The metabolically engineered cell of claim 157,wherein the N-acetylglucosamine b-1,3-galactosyltransferase is aglycosyltransferase having a. PFAM domain PF00535, and i. comprises thepeptide of SEQ ID NO:1, ii. comprises the peptide of SEQ ID NO:2, iii.comprises a polypeptide sequence of any one of SEQ ID NO:3, 4, 5, 6, 7or 8, iv. is a functional homologue, variant or derivative of any one ofSEQ ID NO:3, 4, 5, 6, 7 or 8, having at least 80% overall sequenceidentity to the full-length of any one of the N-acetylglucosamineb-1,3-galactosyltransferase polypeptide with SEQ ID NO:3, 4, 5, 6, 7 or8, and having N-acetylglucosamine b-1,3-galactosyltransferase activity,v. comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive amino acid residues from anyone of SEQ ID NO:3, 4, 5, 6, 7 or 8, and having N-acetylglucosamineb-1,3-galactosyltransferase activity, vi. is a functional fragment ofany one of SEQ ID NO:3, 4, 5, 6, 7 or 8, and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or vii. comprises a polypeptidecomprising an peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NO:3, 4, 5, 6, 7 or 8, andhaving N-acetylglucosamine b-1,3-galactosyltransferase activity, or b.PFAM domain IPR002659, and i. comprises the peptide of SEQ ID NO:9, ii.comprises a polypeptide sequence of any one of SEQ ID NO:10, 11, 12 or13, iii. is a functional homologue, variant or derivative of any one ofSEQ ID NO:10, 11, 12 or 13 having at least 80% overall sequence identityto the full-length of any one of the N-acetylglucosamineb-1,3-galactosyltransferase polypeptide of SEQ ID NO:10, 11, 12 or 13and having N-acetylglucosamine b-1,3-galactosyltransferase activity, oriv. comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive amino acid residues from anyone of SEQ ID NO:10, 11, 12 or 13 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, v. is a functional fragment of anyone of SEQ ID NO:10, 11, 12 or 13 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or vi. comprises a polypeptidecomprising an peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NO:10, 11, 12 or 13 and havingN-acetylglucosamine b-1,3-galactosyltransferase activity.
 160. Themetabolically engineered cell of claim 157, wherein theN-acetylglucosamine b-1,4-galactosyltransferase is a glycosyltransferasehaving a. PFAM domain PF01755, and i. comprises the peptide of SEQ IDNO:14, ii. comprises a polypeptide sequence of any one of SEQ ID NO: 15,16, 17, 18, 19, 20, 21, 22 or 23, iii. is a functional homologue,variant or derivative of any one of SEQ ID NO:15, 16, 17, 18, 19, 20,21, 22 or 23, having at least 80% overall sequence identity to thefull-length of any one of the N-acetylglucosamineb-1,4-galactosyltransferase polypeptide of SEQ ID NO:15, 16, 17, 18, 19,20, 21, 22 or 23, and having N-acetylglucosamineb-1,4-galactosyltransferase activity, iv. comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20consecutive amino acid residues from any one of SEQ ID NO:15, 16, 17,18, 19, 20, 21, 22 or 23, and having N-acetylglucosamineb-1,4-galactosyltransferase activity, v. is a functional fragment of anyone of SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22 or 23, and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or vi.comprises a polypeptide comprising an peptide having at least 80%sequence identity to the full-length peptide of any one of SEQ ID NO:15,16, 17, 18, 19, 20, 21, 22 or 23, and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or b. PFAM domain PF00535, and i.comprises the peptide of SEQ ID NO:24, ii. comprises the peptide of SEQID NO:25, iii. comprises a polypeptide sequence of any one of SEQ IDNO:26, 27 or 28, iv. is a functional homologue, variant or derivative ofany one of SEQ ID NO:26, 27 or 28 having at least 80% overall sequenceidentity to the full-length of any one of the N-acetylglucosamineb-1,4-galactosyltransferase polypeptide with SEQ ID NO:26, 27 or 28 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, v.comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive amino acid residues from any oneof SEQ ID NO:26, 27 or 28 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, vi. is a functional fragment ofany one of SEQ ID NO:26, 27 or 28 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or vii. comprises a polypeptidecomprising an peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NO:26, 27 or 28 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or c. PFAMdomain PF02709 and not having PFAM domain PF00535, and i. comprises thepeptide of SEQ ID NO:29, ii. comprises the peptide of SEQ ID NO:30, iii.comprises a polypeptide sequence of any one of SEQ ID NO:31, 32, 33, 34,35 or 36, iv. is a functional homologue, variant or derivative of anyone of SEQ ID NO:31, 32, 33, 34, 35 or 36 having at least 80% overallsequence identity to the full-length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNO:31, 32, 33, 34, 35 or 36 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or v. comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20consecutive amino acid residues from any one of SEQ ID NO:31, 32, 33,34, 35 or 36 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, vi. is a functional fragment of any one of SEQ ID NO:31, 32,33, 34, 35 or 36 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or vii. comprises a polypeptidecomprising an peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NO:31, 32, 33, 34, 35 or 36 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or d.PFAM domain PF03808, and i. comprises the peptide of SEQ ID NO:37, ii.comprises the peptide of SEQ ID NO:38, iii. comprises a polypeptidesequence of any one of SEQ ID NO:39, 40 or 41, iv. is a functionalhomologue, variant or derivative of any one of SEQ ID NO:39, 40 or 41having at least 80% overall sequence identity to the full-length of anyone of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptidewith SEQ ID NO:39, 40 or 41 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, v. comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20consecutive amino acid residues from any one of SEQ ID NO:39, 40 or 41and having N-acetylglucosamine b-1,4-galactosyltransferase activity, vi.is a functional fragment of any one of SEQ ID NO:39, 40 or 41 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or vii.comprises a polypeptide comprising an peptide having at least 80%sequence identity to the full-length peptide of any one of SEQ ID NO:39,40 or 41 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity.
 161. The metabolically engineered cell of claim 155, whereina. the glucosamine 6-phosphate N-acetyltransferase is a polypeptidesequence comprising the polypeptide of UniProt ID P43577 or is afunctional homologue, variant or derivative of the polypeptide ofUniProt ID P43577 having at least 80% overall sequence identity to thefull-length of the polypeptide of UniProt ID P43577 and havingglucosamine 6-phosphate N-acetyltransferase activity, and b. theL-glutamine-D-fructose-6-phosphate aminotransferase is a polypeptidesequence comprising the polypeptide of UniProt ID P17169 or is afunctional homologue, variant or derivative of the polypeptide ofUniProt ID P17169 having at least 80% overall sequence identity to thefull-length of the polypeptide of UniProt ID P17169 and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity; or is amodified version that differs from the polypeptide of UniProt ID P17169by an A39T, an R250C and an G472S mutation.
 162. The metabolicallyengineered cell of claim 147, wherein the cell is capable ofcatabolizing a carbon source selected from the group consisting ofglucose, fructose, galactose, lactose, sucrose, maltose,malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose,mannose, methanol, ethanol, arabinose, trehalose, starch, cellulose,hemi-cellulose, corn-steep liquor, high-fructose syrup, molasses,glycerol, acetate, citrate, lactate, and pyruvate.
 163. Themetabolically engineered cell of claim 147, wherein the cell is unableto convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate,and/or unable to convert glucosamine-6-phosphate to fructose-6-phoshate.164. The metabolically engineered cell of claim 147, wherein the cell ismodified to produce GDP-fucose, wherein the modification comprises:knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-1-phosphatetransferase encoding gene, over-expression of a GDP-L-fucose synthaseencoding gene, over-expression of a GDP-mannose 4,6-dehydratase encodinggene, over-expression of a mannose-1-phosphate guanylyltransferaseencoding gene, over-expression of a phosphomannomutase encoding gene, orover-expression of a mannose-6-phosphate isomerase encoding gene. 165.The metabolically engineered cell of claim 147, wherein the cell ismodified to produce UDP-galactose, wherein the modification comprises:knock-out of an 5′-nucleotidase/UDP-sugar hydrolase encoding gene orknock-out of a galactose-1-phosphate uridylyltransferase encoding gene.166. The metabolically engineered cell of claim 147, wherein the cell ismodified to produce CMP-N-acetylneuraminic acid, wherein themodification comprises: over-expression of an CMP-sialic acid synthetaseencoding gene, over-expression of a sialate synthase encoding gene, orover-expression of an N-acetyl-D-glucosamine 2-epimerase encoding gene.167. The metabolically engineered cell of claim 147, wherein the cell iscapable of expressing at least one further glycosyltransferase, whereinthe further glycosyltransferase is selected from the group consisting offucosyltransferases, sialyltransferases, galactosyltransferases,glucosyltransferases, mannosyltransferases,N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases,N-acetylmannosaminyltransferases, xylosyltransferases,glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases, N-acetylrhamnosyltransferases,UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases,UDP-N-acetylglucosamine enolpyruvyl transferases, andfucosaminyltransferases, and optionally, the cell is modified in theexpression or activity of the further glycosyltransferase.
 168. Themetabolically engineered cell of claim 147, wherein the cell uses one ormore precursor(s) to produce the disaccharide or oligosaccharide havinga GlcNAc unit at the reducing end, the precursor(s) being fed to thecell from the cultivation medium or the cell produces one or moreprecursor(s) to produce the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end.
 169. The metabolically engineered cellof claim 168, wherein the precursor to produce the disaccharide oroligosaccharide is completely converted into the disaccharide oroligosaccharide having a GlcNAc unit at the reducing end.
 170. Themetabolically engineered cell of claim 147, wherein the cell producesthe disaccharide or oligosaccharide having a GlcNAc unit at the reducingend intracellularly and wherein a fraction or substantially all of theproduced disaccharide or oligosaccharide having a GlcNAc unit at thereducing end remains intracellularly and/or is excreted outside the cellvia passive or active transport.
 171. The metabolically engineered cellof claim 147, wherein the cell expresses a membrane transporter proteinhaving transport activity and transports compounds across an outermembrane of the cell, wherein the cell is modified in the expression oractivity of the membrane transporter protein having transport activity,or the membrane transporter protein having transport activity isselected from the group consisting of a porter,P—P-bond-hydrolysis-driven transporter, β-barrel porin, auxiliarytransport protein, putative transport protein, andphosphotransfer-driven group translocator.
 172. The metabolicallyengineered cell of claim 171, wherein the membrane transporter proteinhaving transport activity: controls the flow over an outer membrane ofthe disaccharide or oligosaccharide having a GlcNAc unit at the reducingend and/or of one or more precursor(s) and/or acceptor(s) to be used inthe production of the disaccharide or oligosaccharide having a GlcNAcunit at the reducing end, and/or provides improved production and/orenabled and/or enhanced efflux of the disaccharide or oligosaccharidehaving a GlcNAc unit at the reducing end.
 173. The metabolicallyengineered cell of claim 147, wherein the cell comprises a modificationfor reduced production of acetate compared to a non-modified progenitor,optionally the cell comprises a lower or reduced expression and/orabolished, impaired, reduced, or delayed activity of at least one of abeta-galactosidase, galactoside O-acetyltransferase,N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphatedeaminase, N-acetylglucosamine repressor, ribonucleotidemonophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase,N-acetylneuraminate lyase, N-acetylmannosamine kinase,N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man,EIID-Man, ushA, galactose-1-phosphate uridylyltransferase,glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase,ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobicrespiration control protein, transcriptional repressor IclR, lonprotease, glucose-specific translocating phosphotransferase enzyme IIBCcomponent ptsG, glucose-specific translocating phosphotransferase (PTS)enzyme IIBC component malX, enzyme IIA^(Glc), beta-glucoside specificPTS enzyme II, fructose-specific PTS multiphosphoryl transfer proteinFruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase,pyruvate-formate lyase, acetate kinase, phosphoacyltransferase,phosphate acetyltransferase, or pyruvate decarboxylase compared to anon-modified progenitor.
 174. The metabolically engineered cell of claim147, wherein the cell is capable of producing phosphoenolpyruvate (PEP)and/or wherein the cell is modified for enhanced production and/orsupply of PEP compared to a non-modified progenitor.
 175. Themetabolically engineered cell of claim 147, wherein the cell comprises acatabolic pathway for selected monosaccharide, disaccharide oroligosaccharide which is at least partially inactivated, themonosaccharide, disaccharide, or oligosaccharide being involved inand/or required to produce the disaccharide or oligosaccharide having aGlcNAc unit at the reducing end.
 176. The metabolically engineered cellof claim 147, wherein the cell resists lactose killing when grown in anenvironment in which lactose is combined with one or more other carbonsource(s).
 177. The metabolically engineered cell of claim 147, whereinthe cell produces 90 g/L or more of the disaccharide or oligosaccharidehaving a GlcNAc at the reducing end in the whole broth and/orsupernatant, and/or wherein the disaccharide or oligosaccharide having aGlcNAc at the reducing end in the whole broth and/or supernatant has apurity of at least 80% measured on the total amount of the disaccharideor oligosaccharide having a GlcNAc unit at the reducing end and itsprecursor(s) in the whole broth and/or supernatant, respectively. 178.The metabolically engineered cell of claim 147, wherein the cellproduces a mixture of charged and/or neutral disaccharides andoligosaccharides comprising at least one disaccharide or oligosaccharidehaving a GlcNAc unit at the reducing end.
 179. The metabolicallyengineered cell of claim 147, wherein the cell produces a mixture ofcharged and/or neutral oligosaccharides comprising at least oneoligosaccharide having a GlcNAc unit at the reducing end.
 180. Themetabolically engineered cell of claim 147, wherein the oligosaccharideis selected from the group consisting of 2-fucosyl lacto-N-biose,4-fucosyl lacto-N-biose, 2-4-difucosyl lacto-N-biose, 3′-sialyllacto-N-biose, 6′-sialyl lacto-N-biose, 3′,6′-disialyl lacto-N-biose,6,6′-disialyl lacto-N-biose, 2′-fucosyl-3′-sialyl lacto-N-biose,2′-fucosyl-6′-sialyl lacto-N-biose, 4-fucosyl-3′-sialyl lacto-N-biose,4-fucosyl-6′-sialyl lacto-N-biose, 2-fucosyl N-acetyllactosamine,3′-fucosyl N-acetyllactosamine, 2,3′-difucosyl N-acetyllactosamine,3′-sialyl N-acetyllactosamine, 6′-sialyl N-acetyllactosamine,3′,6′-disialyl N-acetyllactosamine, 6,6′-disialyl N-acetyllactosamine,2′-fucosyl-3′-sialyl N-acetyllactosamine, 2′-fucosyl-6′-sialylN-acetyllactosamine, 3-fucosyl-3′-sialyl N-acetyllactosamine,3′-fucosyl-6′-sialyl N-acetyllactosamine, P1 trisaccharide(Gal-a1,4-Gal-b1,4-GlcNAc), the xenotransplantation epitope(Gal-a1,3-Gal-b1,4-GlcNAc), Gal-b14-(Galb13)-GlcNAc,poly-N-acetyllactosamine, and GalNAc-b1,3-Gal-b1,4-GlcNAc.
 181. Themetabolically engineered cell of claim 147, wherein the disaccharidehaving a GlcNAc unit at the reducing end does not comprise chitobiose(GlcNAc-GlcNAc).
 182. The metabolically engineered cell of claim 147,wherein the oligosaccharide having a GlcNAc unit at the reducing enddoes not comprise a chitobiose at the reducing end.
 183. Themetabolically engineered cell of claim 147, wherein the cell is abacterium, fungus, yeast, a plant cell, an animal cell, or a protozoancell.
 184. The metabolically engineered cell of claim 183, wherein thecell is a viable Gram-negative bacterium that comprises a reduced orabolished synthesis of poly-N-acetyl-glucosamine (PNAG), enterobacterialcommon antigen (ECA), cellulose, colanic acid, core oligosaccharides,osmoregulated periplasmic glucans (OPG), glucosylglycerol, glycan,and/or trehalose compared to a non-modified progenitor.
 185. The methodaccording to claim 109, wherein the cell is a bacterium, fungus, yeast,a plant cell, an animal cell, or a protozoan cell.
 186. The methodaccording to claim 185, wherein the cell is a viable Gram-negativebacterium that comprises a reduced or abolished synthesis ofpoly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA),cellulose, colanic acid, core oligosaccharides, osmoregulatedperiplasmic glucans (OPG), glucosylglycerol, glycan, and/or trehalosecompared to a non-modified progenitor.
 187. The method according toclaim 109, wherein the separation comprises at least one of thefollowing: clarification, ultrafiltration, nanofiltration, two-phasepartitioning, reverse osmosis, microfiltration, activated charcoal orcarbon treatment, treatment with non-ionic surfactants, enzymaticdigestion, tangential flow high-performance filtration, tangential flowultrafiltration, affinity chromatography, ion exchange chromatography,hydrophobic interaction chromatography, gel filtration, and/or ligandexchange chromatography.
 188. The method according to claim 109, furthercomprising purifying the disaccharide or oligosaccharide from the cell,wherein the purification comprises at least one of the following: use ofactivated charcoal or carbon, use of charcoal, nanofiltration,ultrafiltration, electrophoresis, enzymatic treatment or ion exchange,use of alcohols, use of aqueous alcohol mixtures, crystallization,evaporation, precipitation, drying, spray drying, lyophilization, sprayfreeze drying, freeze spray drying, band drying, belt drying, vacuumband drying, vacuum belt drying, drum drying, roller drying, vacuum drumdrying, or vacuum roller drying.
 189. A method of producing adisaccharide or oligosaccharide having an N-acetylglucosamine unit atthe reducing end, the method comprising: i) cultivating the cell ofclaim 147 in culture medium under conditions permissive to produce thedisaccharide or oligosaccharide having an N-acetylglucosamine unit atthe reducing end, and ii) optionally separating the disaccharide oroligosaccharide having an N-acetylglucosamine unit at the reducing endfrom the cultivation.
 190. A method of producing a sialylated orfucosylated form of lacto-N-biose, the method comprising: i) cultivatingthe cell of claim 147 in culture medium under conditions permissive toproduce the sialylated or fucosylated form of lacto-N-biose, and ii)optionally separating the sialylated or fucosylated form oflacto-N-biose from the cultivation.